Anatomic Pathology / CONSTRUCTING TISSUE MICROARRAYS
Constructing Tissue Microarrays Without Prefabricating
Recipient Blocks
A Novel Approach
Ni Chen, MD, and Qiao Zhou, MD, PhD
Key Words: Tissue microarray; Construction; Technique; Method; Application
DOI: 10.1309/LHCJRFBUH8Q6QD3N
Abstract
Tissue microarray (TMA) is a powerful research
tool and is applied in such diverse areas as tumor
marker validation and laboratory quality control.
Existing TMA construction techniques require an
essential step of prefabricating recipient paraffin blocks
on which holes are punched so that tissue cores can be
inserted. This procedure has several disadvantages,
such as accidental block breakage during hole
punching and difficulty ensuring that the cores are flush
with the block surface. We developed a novel TMA
construction technique without prefabricating recipient
blocks. We used double-sided adhesive tape attached to
x-ray film as an adhesive platform on which the tissue
cores were placed securely. The array of tissue cores
then was embedded in an embedding mold. We have
been making high-quality TMAs with up to 220 cores
within 2 to 3 hours using this highly dependable,
efficient, versatile, and cost-effective technique, which
can be adopted by pathology laboratories and
researchers with minimal investment.
Tissue microarray (TMA) technology has emerged as a
powerful tool in biomedical research,1-4 finding use in such
diverse areas as tumor marker validation and expression profiling,1-8 molecular classification and prognostication of
tumors,7,9-12 and interlaboratory and intralaboratory quality
control,13-17 among other applications.18-20 Together with
DNA microarrays and emerging protein and antibody
microarrays, TMA is considered one of the essential tools in
large-scale, high-throughput analysis.18-20 Whereas DNA
and protein microarrays aim at simultaneous assay of hundreds to tens of thousands of genes or proteins in a cell or tissue sample, TMA is the ideal approach to assaying, for
example, the expression status of a gene (eg, by in situ
hybridization) or protein (eg, by immunohistochemical
analysis) in up to hundreds of samples simultaneously.18-20
In principle, the construction of a TMA consists of transferring tissue cores from various donor blocks to a recipient
block, which then is used to cut sections containing all the tissue cores embedded in the recipient block.1-4 Techniques
described in the literature1-4,21 use an essential step of prefabricating recipient paraffin blocks on which holes are punched
so that tissue cores can be inserted.
This approach has several disadvantages. First, block
breakage during hole punching will result in an unusable
recipient block. This is particularly serious in procedures in
which hole punching and core insertion were performed one
by one, because breakage of a half-finished recipient block
with dozens of tissues cores is difficult to handle and might
result in waste of precious tissue samples. Second, for manual techniques that are adopted by many laboratories, it is difficult to punch well-aligned and closely packed holes. Third,
tissue cores, which frequently are not of the same length, can
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hardly be placed in the recipient block so that all cores always
are flush with the surface. Thus, to cut sections carrying all
the cores, it often is necessary to trim the blocks, resulting in
loss of valuable tissue samples. Fourth, any tiny space
between the cores and the paraffin around them, despite a
paraffin filling step, might adversely influence appropriate
merging of the two during later procedures, resulting in
unsatisfactory blocks and sections.
We developed an apparently simple, yet highly dependable, efficient, and adaptable technique of TMA construction
based on an adhesive platform without prefabricating recipient blocks, which requires no special instruments and, thus,
little investment. We believe this novel approach to TMA construction will be useful and applicable in all laboratories interested in TMA construction.
Materials and Methods
Materials and Instruments
A 1-mm diameter core biopsy needle with stylet
(Shanghai SA Medical & Plastic Instruments, Shanghai,
China) was used for coring ❚Image 1A❚. Needles of different gauge could be used according to researchers’ needs.
The needle was blunted. Double-sided adhesive tape, a pair
of stainless steel tweezers with curved tips, razor blades,
discarded x-ray film, and embedding molds (such as the
one from Leica, Heidelberg, Germany, shown in ❚Image
1C❚ or any similar molds) were the basic materials needed
(Image 1).
A
B
Locating the Coring Site
The H&E-stained sections from donor blocks were
reviewed and coring site marked on the block according to
H&E histologic findings.
Preparing the Adhesive Platform
A piece of x-ray film (or similar supporting material) was
cut according to the size of the embedding mold. The piece we
used was 3.5 × 2.5 cm. A piece of double-sided adhesive tape
was cut to size and attached to the x-ray film, and the protective paper on top of the adhesive tape was removed to expose
the adhesive surface. The platform was now ready to receive
tissue cores. Alternatively, to help align the tissue cores, various formats of lanes could be created by cutting with a razor
blade and removing strips of the protective paper, as shown in
❚Image 2❚.
Retrieving Tissue Cores
The core biopsy needle was drilled 3 to 4 mm into the
donor block at the prelocated site. After pulling the needle out,
the stylet was inserted to carefully push out the tissue core,
which could be placed temporarily in a well of a 96-well plate,
in the planned order of placement according to array layout to
avoid errors, if it was not attached immediately to the adhesive
tape platform.
Arraying the Tissue Cores
Each retrieved tissue core was trimmed minimally at
one end with a sharp razor blade to make the end flat and
wax-free. The tissue core then was attached securely on the
adhesive platform by using the tweezers according to the
C
❚Image 1❚ A, Basic materials for tissue microarray construction without prefabricating recipient blocks: a modified core biopsy
needle with stylet, donor blocks (shown with holes resulting from coring), a pair of tweezers for handling the cores, and an
optional tissue cassette cover (to help align the cores as a module when attaching the tissue cores to the tape). Also shown is a
finished platform with tissue cores in place and a close-up view (B). The tissue cores are attached securely to the adhesive
platform, resembling columns on a plate. C, The platform is fit into an embedding mold, ready for embedding.
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planned layout. The completed array looks like columns of
tissue cores standing on their flat ends on the platform ❚Image
1B❚. A tissue cassette cover (such as the yellow cover shown
in Image 1A or any similar covers) could be used optionally
to help align the cores by laying the cassette cover on top of
the adhesive platform, which resulted in the 7 × 9 array
shown in Image 1B. The alternative and preferred approach
of creating lanes, as shown in Image 2, could be used to construct other layouts. Moreover, using this lane-based platform
also helped to avoid tissue core tipping during embedding,
and it facilitated peeling the adhesive tape from the paraffin
block after embedding.
Embedding
Another piece of double-sided adhesive tape was applied
to the bottom of the x-ray film, and the adhesive platform
with tissue cores in situ was placed into an embedding mold
A
C
(Image 1C), which then was preheated briefly at 60°C on the
heat plate of an embedding station (eg, Leica EG 1140,
Leica) or, alternatively, in an oven, just to slightly soften the
tissue cores. Paraffin at approximately 70°C was injected
carefully to fill the mold and then cooled to room temperature. Arrays with higher core density (usually >100 cores)
were placed on the heat plate briefly to achieve complete
merging of the tissue cores with injected paraffin before cooling to room temperature.
Finishing the Block
The block was cooled further at 4°C for 10 minutes.
The x-ray film and the adhesive tape at the bottom of
the block were peeled off. The exposed surface was the
cutting surface ready for sectioning, with all tissue
cores flush with the block surface, and minimal trimming was needed.
B
❚Image 2❚ More examples of array platforms under construction
to show various formats that can be adopted. A, 6 × 16 array.
B, Two sectors of a 5 × 10 array. C, An H&E-stained section (7
× 13 array). Alignment was facilitated by creating lanes on the
adhesive platform (A and B, viewed from top of the platform).
This preferred alternative circumvented the restrictions
imposed when using the cassette cover, and offered more
closely packed cores, less intercore spacing, and superior
alignment of the cores.
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Results and Discussion
The procedures (Images 1 and 2) and a TMA block
❚Image 3❚ made by this technique are illustrated, as are H&Estained (Images 2C and 3) ❚Image 4A❚ and immunostained
❚Image 4B❚ sections from the block. Although we illustrated
the procedure with 7 × 9 arrays, other formats can be adopted
easily as desired (Image 2). We have been routinely making
60- to 220-core TMAs by this technique. It is desirable to
❚Image 3❚ Finished tissue microarray (TMA) block and
sections (H&E-stained) made from the block (left). Close-up of
a TMA section, illustrating the quality of the cores (right). Use
of the cassette cover to line up the core resulted in wider but
even spacing between the cores.
A
divide the cores into sectors (as shown in Image 2B). Each
laboratory may use a format that best fits its needs.
The method we describe is highly dependable. Because
the cutting surface is flat with tissue cores flush with the block
surface when embedding is finished, minimal trimming is
needed before sectioning. TMAs with 3-mm-long tissue cores
typically can cut hundreds of 4-µm serial sections with all
cores represented, and only a few core losses or displacements
will be encountered. Thus, rare and valuable tissue samples
are conserved more effectively. If donor blocks with thicker
tissue chunks were secured, a much larger number of sections
could be obtained.
The technique also is very efficient. We are able to routinely make a 100-core TMA within just 2 hours, starting from
coring and ending with finishing the blocks. Personnel training also is minimal, and a technician can learn the procedure
essentially in 1 day.
The technique we describe is straightforward, and we
have encountered few problems. Although it is conceivable
that tissue cores could be broken during handling, we have not
encountered this problem during transferring from the donor
block to the adhesive platform or during embedding.
Nevertheless, it is advisable to pay attention to the following
points to obtain the best results with the technique: (1)
Planning of the layout is essential before coring and arraying.
Core-core spacing must be considered before starting. (2)
Before the cores are placed onto the adhesive platform, make
sure one end of the core is flat so that it will stand vertically
on end. (3) This end also should be essentially wax-free to be
attached securely to the tape. The presence of paraffin at the
core end could result in tipping of cores during embedding as
B
❚Image 4❚ A, A representative tissue core of malignant melanoma (H&E). B, Streptavidin-alkaline phosphatase immunostaining
for HMB45 (DAKO, Carpinteria, CA) in a pigmented malignant melanoma. Visualization was with AP red (Zymed, South San
Francisco, CA), resulting in positive signals of red contrasting well with the melanin pigment.
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paraffin melts, although this can be remedied by using the
tweezers. (4) The mold preheating step (brief preheating at
60°C before injecting 70°C paraffin, just to slightly soften the
cores) was very helpful to achieve superb merging of the
injected paraffin and the tissue cores, preventing any tiny
space that could result from imperfect merging and consequent sectioning problems. However, it is important to avoid
overheating or prolonged heating because this could result in
core tipping. (5) On the embedding station, after the preheating step, the embedding temperature of approximately 70°C
generally gives superior results. To achieve complete merging
of the tissue cores and the injected paraffin, which is vital for
high-quality array blocks, as well as sections, arrays with
higher tissue core density might need to be heated briefly on
the heat plate after paraffin injection and then cooled to room
temperature.
This novel TMA construction method, which is versatile,
dependable, efficient, and cost-effective, offers numerous
advantages and avoids problems associated with procedures
that require prefabricating recipient blocks. Although we
described a manual procedure, instrumentation based on the
principles is conceivable.
From the Pathology Department, West China Hospital, West China
Medical School, Sichuan University, Chengdu, China.
Supported in part by grant 30125023 from the Natural
Science Funding Committee and grant 2002CCA01400 from the
Ministry of Science and Technology of China, Beijing.
Address reprint requests to Dr Zhou: Pathology Dept, West
China Hospital, West China Medical School, Sichuan University,
Chengdu, 610041, China.
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