Genomic approaches such as RNA profiling are providing a new powerful means to discover
disease-related genes. One of the most challenging aspects presented by high throughput gene expression
approaches is that they usually generate a large battery of potential targets. Determination of which
genes are truly important for classification in terms of diagnosis, prognosis, and therapy represents a
bottleneck. How does one begin to validate and prioritize potential targets? Often the first step in
attempts to discover the disease relevance of a given gene is the elucidation of the precise cells that
express it in normal and diseased human tissues. This is even more powerful if it can be done
simultaneously with an assessment of clinical significance. A major limitation, however, is that in situ
based molecular analysis is cumbersome and often limited by the availability of suitable reagents such as
high quality antibodies or a robust system for in situ hybridization. In addition, adequate validation
of biomarker expression often requires large patient cohorts with long-term clinical follow-up. Finally,
interpretation of expression results requires a pathologist. While many of these limitations have yet to
be solved, Tissue Microarrays (TMAs) are emerging as a breakthrough in our ability to rapidly analyze the
expression of existing and new biomarkers using archival pathology specimens.
Multi-tissue blocks were first introduced by Battifora et al. in the so-called "sausage" or
Multi-Tissue Tumor Block (MTTB) where up to 100 separate tissues were processed together into a single
paraffin block . Recently, Kononen et al. introduced a new method of combining multiple
tissues into a single paraffin block that uses a novel sampling approach, with regular size and shaped
tissues. This allows for many more specimens that are precisely arrayed to be inserted into the
blocks ; for reviews see
What is a Tissue Microarray and What are its Advantages Over Standard Tissue Blocks?
The TMA consists of cylindrical paraffin embedded tissue cores that are acquired from
primary "donor" blocks. The donor block is a standard tissue block that may be from surgical pathology,
autopsy or research material. A morphologically representative area of interest within the donor block
is identified under the microscope using a stained section (usually Hematoxylin and Eosin stained) on a
glass slide as a guide. The tissue cores are removed from the donor and inserted into a "recipient"
paraffin block using a custom patented instrument from Beecher Instruments. Using a precise spacing
pattern, tissues are inserted at high density, with up to 1000 tissue cores in a single paraffin block.
Sections from this block that are cut with a microtome are placed onto standard slides that can then be
used for in situ analysis. Depending on the overall depth of tissue remaining in the donor blocks,
tissue arrays can generate between 100 and 500 sections. Once constructed tissue microarrays can be used
with a wide range of techniques including histochemical staining, immunohistochemical/immunofluorescent
staining, or in situ hybridization for either DNA or mRNA.
Figure 1. Example of prostate TMA containing 400 tissue cores (20 cores x 20 cores, 0.6 mm each). Immunohistochemical staining for alpha methyl acyl CoA racemase (AMACR). A low power view (upper left) shows the entire array. We typically place a column of assorted control tissues in the 9th column and 3 additional columns of assorted control tissues in columns 18-20. A medium power is shown in the upper right that corresponds to the boxed area from the low power view. A higher power view is shown below that corresponds to the boxed area in the medium power view. This shows strong staining in prostate adenocarcinoma and weak/negative staining in normal prostate epithelium. Images were obtained with the Bacus Labs BLISS Imaging Workstation.
Since relatively small areas of tissue (down to 0.6 mm in diameter) are obtained from the donor
blocks, this method can help to expand the usefulness of existing archival paraffin blocks by
facilitating the construction of multiple "duplicate" blocks. This significantly expands the capacity of
the tissue samples, indicating that more studies can be performed using limited samples. Other
advantages of TMAs are that they are designed for high throughput screening of expression, while
providing uniform reaction conditions and multiple built-in potential positive and negative controls.
Since only one or a few slides are subjected to the staining procedure, TMAs also allows one to economize
use of reagents, which can at times be quite limiting. Since several hundred cases are now present on
one or few slides, TMAs also cut down on microtome sectioning of numerous paraffin blocks. It should be
pointed out that even after removal of cores from donor blocks, these donor blocks usually still retain
sufficient residual tissue for adequate pathological interpretation.
Are Tissue Microarrays Valid for Clinico-Pathological Studies?
The most frequently asked question regarding tissue microarrays is how do they account for
heterogeneity of tissues? Camp et al., examined the number of "disks" or TMA spots required to
adequately represent the expression of three common antigens, estrogen receptor (ER), progesterone
receptor (PR) and the Her2/neu oncogene, in 38 cases of invasive breast carcinoma . They made
TMAs containing 10 tissue cores from the same tumor and compared the results of analysis of staining of
the TMA cores to that obtained using a single standard whole tissue section from which the TMA cores were
derived. They found that two spots produced similar results to the whole tissue in more than 95% of the
The largest published study to date to address the issue of tissue heterogeneity is that of Torhorst
et al. who examined ER, PR and p53 in breast carcinoma  . In this study, the clinical
relevance of staining was examined by comparing immunostaining results using standard sections versus TMA
slides in 553 breast cancer patients. Four high-density TMA blocks were constructed, each containing 1
core from a different region of the tumor from each patient. For ER, the range of positive staining from
the 4 different blocks was from 78.9 to 80.8%, which compares to that observed in a large standard
section (79.8%). When using a single 0.6 mm sample, loss of ER correlated inversely with disease
specific survival to a similar extent as standard sections. Thus, very little benefit was obtained using
more than one spot for ER. For PR, the addition of multiple spots analyzed increased the frequency of
positive staining towards that obtained with standard sections (41.1% with 1 spot, 53.1% with 4 spots,
compared with 60.3% with conventional sections). Loss of PR as analyzed on TMA spots was also predictive
of poor outcome in a manner similar to that of the conventional section, even when only 1 0.6 mm core was
used. For p53, the frequency of positive staining was less using TMAs than when using standard slides
(15.2-20.9% for single spots, up to 24.1% for all 4 combined, as compared to 42.8% for conventional
sections). However, in terms of prognostic significance the correlation between p53 staining and poor
outcome was stronger using TMA spots, even one spot, than was that of conventional sections.
In terms of prostate cancer, Rubin et al. used digital image analysis (CAS200, Bacus Labs, Lombard,
IL) on TMAs containing 10 replicate tumor samples from 88 cases of prostate cancer  to
evaluate Ki-67 expression for each case. Four cores provided the optimal sampling for TMA cores using a
Cox proportional hazards analysis to determine predictors of time until PSA recurrence following radical
prostatectomy for clinically localized prostate cancer. Fewer TMA samples significantly increased Ki-67
variability and a larger number did not significantly improve accuracy.
Several other studies have also examined the question of the representation of tissues in TMAs using
various markers in different tumor types
. In general, although they vary somewhat in
terms of the recommendations for sampling, all studies indicate there is usually excellent agreement
between the use of TMAs and standard tissue sections for clinico-pathological studies.
From a theoretical point of view the question of how many samples are required to adequately perform
a study is related to the variability of the parameter being analyzed. Thus, for homogeneous markers, a
single TMA spot per case will be adequate. At our institution we routinely take 4 cores each from areas
of prostate tumors and matched normal tissue (Fig. 1) in order to maximize the usefulness of the TMA
since we do not know what biomarkers we will be applying in the future. Thus for some studies, this will
be "overkill" and for others it may be barely adequate.
Another potential difficulty in terms of how many cores to take involves the fact that not all TMA
cores will be present on all TMA slides. Having at least one additional TMA core will help ensure the
presence of the number of cores that one hopes to obtain on the final TMA slide.
Digital Image Acquisition and Analysis
TMA slides can be viewed under conventional microscopes. In this case a key, usually in the form of a
spreadsheet, corresponding to the x y coordinate system is used and histopathological diagnoses and
interpretations are recorded. The data can be recorded on paper for later entry into a spreadsheet or
database, or, it can be entered directly into the computer. One of the difficulties with this approach
is that since the array spots do not have their coordinates printed on the slide it is likely that the
user may loose track of the x and y coordinates of given spots and have to repeatedly become reoriented.
Prostate SPORE Approach to Imaging
Several groups have been developing methods to acquire and archive digital images of the TMA spots for
evaluation on a computer monitor such that the data is linked to underlying clinico-pathological
information regarding the array spot. To acquire digital images of TMA spots a number of users have been
using the Bacus Labs Incorporated Slide Scanner (BLISS, Bacus™
laboratories, Lombard, IL)  as previously described
. The BLISS imaging
system consists of a Zeiss microscope equipped with a software driven motorized stage, integrated digital
video camera, and a customized personal computer running Microsoft Windows. The Bacus Labs Tracer
software program is designed to scan entire glass microscope slides, or any part of the slide, using any
available microscope objective. The slides can then be viewed using the free downloadable WebSlide®
Viewer. Slide images are generally stored on a server running WebSlide Server software from Bacus Labs.
Figure 2. Example of TMA spot from TMAJ software program. Image were obtained with the Bacus Labs BLISS Imaging Workstation.
The system has been adapted to scan TMA spots using customizable features that were developed in
collaboration with the Prostate SPORE Tissue Microarray Working Group (University of Michigan, The Dana
Farber Cancer Institute, Baylor College of Medicine, and Johns Hopkins University School of
. The operator then indicates through the software the number and location of
the array spots. All array spots are then automatically scanned at full resolution (in most cases a 20x
Zeiss Plan-Apochromat ® objective is used, although other objectives can be used). Each array spot is
imaged as 6 individual 640 x 480 pixel images that the software automatically "tiles" into a single
composite image. The composite images are stored in a file containing the embedded x y coordinates from
the tissue array spot, along with user provided information regarding the TMA slide that was scanned.
The composite image files can be viewed individually using any number of image viewers, or can be
imported into a relational database and related by their x y coordinates to the specimen from which they
were derived (Fig. 2). More recently, Bacus Labs have developed an Active X plug-in that is designed to
facilitate image handling for viewing TMA spots directly from inside a database application of your
choosing. Several other systems that are either being adapted or are potentially adaptable to imaging
TMA spots are shown in Table 1. In our laboratory we have begun to successfully employ the Chromavision
ACIS II for scanning whole TMA slides .
Table 1. Digital microscopic imaging systems that already adapted for TMAs, are
in the process of adapting, or may be adaptable for TMA scanning.
TMA Clinico-Pathological and Image Data
TMA-based technology prompted the need for a system that effectively managed data generated from this
high-throughput approach. The use of a large spreadsheet has been the standard solution to handle
voluminous amounts of data. This approach is useful for experiments on a one-time basis, but becomes
very cumbersome when analyzing multiple markers on a given specimen or when having multiple observers
render diagnoses and scores on a given specimen. As part of the same on-going collaborations between the
Specialized Programs in Research Excellence for prostate cancer, the three groups from the University of
Michigan, Johns Hopkins University, and Baylor College of Medicine (Houston, TX) have been developing
systems to manage TMA clinical data and TMA image data
. The overall architecture of the
system is that the TMA images are examined on a computer screen where the image is presented within a
database form. The user then enters data regarding the image directly into the form. The type of data
that is recorded is flexible and can include items such as image quality, diagnosis, and
immunohistochemical scoring. An example of one such database form is shown in Fig. 2. A demonstration
edition of this software, referred to as TMA-J can be found at (http://tmaj.pathology.jhmi.edu).
Another system, also based on scanning TMA spots using the BLISS system, has been presented by the
Stanford group . The approach was to develop two software programs to aid in analysis of
staining results and rapid retrieval of TMA spot digital images. The authors designed a program that
allows for rapid transformation of immunostained data, recorded in Microsoft Excel, into a format that
can be used for cluster analysis. The program Cluster is used to group data with regards to tumor
staining pattern with antibodies, analogous to tumors grouped according to RNA expression. The Clustered
pattern can be viewed in another freely available software program called Treeview. For an online
demonstration, see (http://genome-www.stanford.edu/TMA/explore.shtml).
At Yale University David L. Rimm's group has developed software that uses a custom imaging microscope
system for scanning TMA slides that are stained with fluorescent markers . Trotter et al.
have presented at this meeting last year on mapping, navigation and data management of TMA
Quantification of Immunohistochemical Staining Using TMASpecial Array Types
At present modules for quantification of TMA results using the Bacus system are under development. A
commercial system for imaging TMA slides has been developed by Chromavision as an extension of their
Automated Cellular Imaging System (ACIS). The system is designed specifically for quantification of
immunohistochemical staining using images obtained by light microscopy. Users can select what type of
data to obtain such as average brown intensity, brown area, etc. The data can then be exported to a
spreadsheet where the individual TMA spots are identified based on their array coordinates. We have
begun to use this system and to import the resulting quantitative data into the TMAJ
database . In terms of academic institutions, Camp et al., at Yale University's TMA Lab
developed a dedicated system for quantifying fluorescent TMA spot images called Aqua . The system uses multiple colors with fluorescent imaging to
automatically quantify staining at the sub-cellular level. This is a particularly intriguing approach
since it is being designed to eliminate the need for the pathologist to interpret each array spot.
At least two groups have so far produced TMAs using frozen tissues
. The advantage to
having frozen tissue arrays is that post fixation can be tightly controlled, some antibodies do not bind
to formalin fixed epitopes, and the quality of nucleic acids (DNA/RNA) is generally much higher. Hoos
and Cordon-Cardo have developed a simple devise, independent of the Beecher Instruments devise for frozen
TMA construction  and Fejzon have adapted the Beecher Instrument machine using dry ice to
keep the donor and recipient blocks frozen .
One of the most powerful types of controls for
immunohistochemical staining and for in situ hybridization is the use of well-defined cell lines. For
example, when one is working up a new antibody against the retinoblastoma protein (pRB), an excellent
negative control would be a retinoblastoma cell line that was shown to be genetically null for RB alleles. Similar approaches are useful for p53, etc. In fact, when one knows
the status of either the genomic DNA corresponding to a given gene in a given cell line, or information
about the expression at the mRNA and/or protein level, then suitable positive and negative controls can
be obtained for immunohistochemistry or in situ hybridization for essentially all non-house keeping
genes. Along these lines, a very useful type of control is to use an isogenic system to induce
expression of a given gene in a cell line that does not normally express it. In this case the untreated
cell serves as the negative control and the treated cell serves as the positive control. We recently
used this approach to develop controls for examining COX-2 expression in prostate cancer cells where we
induced expression of COX-2 using phorbol ester in PC3 cells . We have been isolating cells
grown in culture, fixing them in formalin, embedding them in agarose, and then submitting them for
routine processing into paraffin  (see below for detailed method) The advantage of a
solid-like gel suspension such as agarose is that the cells are not lost in processing, which often
happens when preparing paraffin blocks from cell pellets. A manuscript comparing methods of embedding
cells in culture for TMA production has recently been published .
Government and Commercially Available TMA
For those seeking to obtain slides from existing tissue arrays, there is a NIH program
called Tissue Array Research Program (TARP)
where individual slides are available for purchase at very reasonable rates .
There are also several commercial sources including Invitrogen
Arrays) ,Zymed Laboratories (MaxArray™) ; and SuperBioChips .
Tissue Fixation Issues
One of the most important issues in constructing tissue microarrays is to be sure of the quality of
the tissues used. While there is no best fixative for all types of applications, the vast majority of
archival specimens have been fixed in 10% neutral buffered formalin, which is actually contains 4%
formaldehyde among other chemicals. While many antibodies produce excellent staining using formalin
fixed tissues, not all tissues are properly fixed after "routine fixation". We have found for
p27 [Kip1] that longer fixation times yield more reliable results . For construction
of our prostate TMAs we typically only use tissues that we are certain of the quality of fixation; we use
either tissues that were freshly harvested and sectioned into thin portions (less than or equal to 3 mm
thick) that are fixed in large volumes, or those where the prostates have been injected with formalin to
provide uniform fixative coverage . In addition, all blocks are subjected to
immunohistochemical staining prior to selection for a TMA.
New Target Validation
Our approach at John Hopkins to new target identification and validation has been to discover genes
that are highly over expressed in prostate cancer and attempt to validate expression using
TMAs . In collaboration with William Isaacs and Jun Luo and with the NIH (Jeff Trent's
group), a list of candidate biomarkers was generated from this approach, and we have begun to validate
these candidate markers . Several groups are taking this exciting approach (e.g.
Other Potential Type of TMAs
The types of tissues one can use for construction of TMAs is unlimited. For example those consisting
of human xenograft tumors may greatly extend the ability of many different investigators to have access
to these tissues. In addition, animal tissues as well as xenografts can be subjected to drug treatments
in vivo and then the pattern of gene expression alterations can be documented using cDNA arrays and/or
TMAs. Similar types of studies with human tissue can be obtained and tissues can be arrayed before and
after treatment. The number of cell lines is also growing rapidly and a resource that provides TMAs
containing many cell lines would be very valuable.
Practical Methods of TMA Construction
The following is largely derived from a book chapter that will appear in the Series: Practical
Methods In Molecular Biology by Humana Press.
Several sources of information are available for tissue microarray protocols, tips techniques, and
trouble-shooting. These include recent reviews
, a detailed web site with protocols (http://www.yalepath.org/DEPT/research/YCCTMA/YTMA_protocol.pdf),
and sites developed by the NIH (http://resresources.nci.nih.gov/tarp/; http://www.nhgri.nih.gov/DIR/CGB/TMA.
Tissue microarray construction entails several key aspects that
will be presented as an overview here, with details found in the methods section. These aspects include
the following: 1) purpose of the TMA; 2) TMA design; 3) selection of appropriate tissue blocks; 4)
identifying regions of interest within donor tissue blocks; 5) data handling; 6) array construction; 7)
TMA block sectioning; and 8) TMA slide staining. We present detailed methods of TMA construction below,
using our experience constructing more than 190 TMAs containing more than 30,000 tissue cores as a guide.
In depth information regarding array construction and troubleshooting is also available form the Beecher
Instruments web site (http://www.beecherinstruments.com/index.html) and instruction manual.
Purpose of the TMA
There are unlimited types of TMAs that may be of use depending upon the project at hand.
We have found that very useful arrays can be constructed from an assortment of normal human tissues
obtained as discarded material from surgical specimens. These arrays are ideal for working up new
antibodies or new probes for in situ hybridization. A second general type
of array is one with a small number of diseased tissues from several patients. For example, when
evaluating a new antibody for prostate cancer we often use an array with 20 cases of tumor and normal to
quickly assess whether the marker appears of interest. A more sophisticated TMA can be constructed
containing samples from many more patients (hundreds or more), and this may consist of a set of several
individual TMA blocks. The true power of this type of approach is to correlate staining results with
clinical outcome, if available. If possible, it is always advisable to include some matched normal
tissue from which the tumor or other diseased tissue is derived. For our prostate cancer arrays, we
provide a matched normal appearing epithelial sample from each sample of cancer.
Depending upon the purpose of the TMA the design may vary greatly. There are no established
guidelines for TMA design. For pancreas cancer, where the prediction of clinical outcome is not often an
important issue, the main purpose in constructing a TMA often is to simply assemble a convenient series
of patients into one or few TMA blocks. For example, we have produced arrays of invasive pancreas
adenocarcinoma, pancreatic intraepithelial neoplasia, intraductal pancreatic mucinous neoplasms, etc.
These arrays typically contain between 50 and 100 patient samples.
One key consideration is the size of the punch. There are four sizes available: 0.6 mm, 1.0 mm, 1.5
mm, and 2.0 mm. While 2.0 mm is the preferred size of some investigators, these large cores may damage
the donor and recipient blocks. If larger cores are desired, the 1.5 mm core may be acceptable since it
seems to cause less damage but still covers a large area. The 1.5 mm core is the preferred core size of
most of the pancreas cancer arrays that have been constructed at our institution, since pancreas cancer
can be so sclerotic and tumor cells are often widely dispersed. For the majority of TMAs in our
laboratory, however, the 0.6 mm is used, since many studies have shown that 1-4 cores of this size from a
given tissue yield as much information as standard tissue sections
. The number of cores
that can be put into a single TMA block is another important consideration. The reported record number
of cores is 1200. But it is usual not to place more than 600 (See note [i]).
The spacing between samples is the next consideration. Spaces from 0.1 mm on up have been used
successfully (see note [ii]). Table 2 shows a convenient
spacing pattern between cores and the number of cores that can be put into the array block without
rotating the block during construction.
The array is usually laid out in an electronic spreadsheet. Fig. 3 shows a sample "Map"
used to design and construct an example array utilizing the 0.6 mm punch. The resulting array will have
10 cores across (X-axis) and 6 cores down (Y-axis). This TMA is designed to have four replicate cores
from each of the sample areas (individual tissue diagnoses), with the tumor and normal tissues from each
case in the same row. All pertinent information that identifies a sample should be included in the map
In most of our TMAs we place control normal tissues or cell lines in strategic regions throughout the
blocks. Any kind of control tissue may be used, normal tissues, cell lines, or animal tissue (Note [iii]). Often, we place entire columns of various control
tissues between the tumor and normal tissue, or asymmetrically at one end of the block. It is quite
important to never construct an array block with complete symmetry, as one can loose orientation.
Navigating a stained TMA slide can be cumbersome in that it is easy to loose one's place regarding the
X and Y coordinates. Building in an orientation marker that indicates where the array begins can be very
helpful. As first shown to our group by the M.A. Rubin laboratory (now at Harvard University), we use 5
tissue cores arranged in a plus sign "+", three cores placed vertically intersecting three cores placed
horizontally. This is positioned in the upper left hand corner of the block. Although many TMA labs
prefer to group array cores in separate regions of the blocks, we do not do this since this can hamper
automated image acquisition, at least on some systems.
The selection and collection of tissue blocks to be included in TMAs is the most time consuming aspect
of the entire project. Appropriate samples from clinical specimens are identified from the pathology
archives of the given institution. Glass slides corresponding to the entire cases for surgical pathology
specimens are retrieved and reviewed to select appropriate candidate blocks. Corresponding blocks are
then obtained from the tissue archives. The area to be sampled, which usually represents a region
corresponding to a specific pathological diagnosis ("tissue diagnosis")  or relevant normal
tissue, is circled with a Pilot Pen (or similar xylene free pen) by a pathologist or highly trained
technician. Since the block may contain more than one tissue diagnosis of interest for obtaining tissue
cores for a TMA, each circled tissue diagnosis on the slide is also assigned a letter or number.
Details of array construction and TMA block sectioning are presented below. We recommend that you
familiarize yourself with the equipment by constructing a practice array. The first couple of array
blocks made will be far from perfect. By the third, expert status should be achieved.
TMA Block Sectioning
Sectioning an array can be accomplished using standard sectioning procedures, although it is highly
recommended to use a dedicated microtome. Prior to use the recipient blocks are faced off on this same
microtome. In this fashion the amount of realignment of the microtome block holder is minimized. All
the array blocks produced
Figure 3. Example of spreadsheet containing TMA core data
in the lab will have the same orientation; maximizing the number of slides that an array will yield.
Changing blades frequently is advisable. Several investigators use the "Paraffin Sectioning Aid System"
from Instrumedics Inc. This is called the Tape Transfer Method. Since we are still in the process of
perfecting this method, we refer the reader to the Instrumedics Web site (http://www.instrumedics.com).
TMA Slide Staining
TMA slides can be stained with any stain that can be used for standard slides, with out any
Table 2. Typical core spacing and number of cores using various needle sizes.
|Needle Size ||Spacing Between samples ||Array Format ||Total number of cores|
|0.6mm ||0.2mm ||20X20 cores ||400|
|1.0mm ||0.3mm ||16X13 cores ||208|
|1.5mm ||0.4mm ||11X9 cores ||99|
- Manual Tissue Arrayer (Beecher
Instruments, Silver Spring MD)
- Tissue Array Punches, sizes 0.6mm, 1.0mm, 1.5mm
(Beecher Instruments, Silver Spring MD)
- Paraplast X-tra (Fisher Scientific,
Suwanee, GA Cat # 23-021-401)
- Cap Gap Slides (Fisher Scientific, Suwanee, GA Cat
- Oven (Fisher Scientific, Suwanee, GA Cat #11-695-1)
- Magnifier on Stand with Attached Light (Fisher Scientific, Suwanee, GA Cat # 8-882)
- Stainless Steel Molds 6mm (Allegiance, Columbia, MD Cat #M7300-4)
- Tissue Cassettes (Allegiance, Columbia MD, Cat # TN045)
- Floatation Water Bath (Allegiance, Columbia MD Cat #M7654-1)
- Accu Edge Blades (Allegiance, Columbia, MD Cat # M7321-41)
Pen Ultra Fine Point (Register Office Supply, Baltimore MD Cat #"s 44104, 44103 and 44102 Red, Blue and
- Automated Rotary Microtome (Leica, Deerfield, IL Cat # RM2155)
Although a semi-automated tissue microarrayer is now available from Beecher Instruments, the methods
presented here all involve the manual array system
Tissue selection and donor block preparation
- Identify the tissue of choice using a
standard tissue section on a glass slide stained with hematoxylin and eosin (H&E). An immunostained
slide may also be employed.
- Circle the area to be sampled directly on the glass
slide, which usually represents a region corresponding to a specific pathological diagnosis ("tissue
diagnosis")  or relevant normal tissue, with a Pilot Pen (or similar xylene free pen).
Assign each separate region (tissue diagnosis) its own number or letter, such that it can be uniquely
identified given the case number, block designation and tissue diagnosis designation.
- Over-lay the circled H&E glass slide and the area of interest and identify the
corresponding region on the block and circle with a laboratory marker (see Note [iv]).
- The wax is melted at 56 to 59 °C and then
poured into a deep mold. A standard tissue cassette is placed on top (see Note [v]).
- Allow the block to chill completely on a
cold plate or ice. After cooling, separate the cassette and mold (see Note [vi]).
Equipment Set Up For TMA Construction
- Each block and slide pair should be
arranged in the order that they will be used following the map design.
the recipient block into the base plate and position the base plate on the arrayer.
- Position a pair of punches in the arrayer. The larger one is used for extracting tissue
from the donor block and is placed in the right punch holder (for right handed persons). The smaller one
is for making a hole in the recipient block where the donor tissue will be positioned, and is placed in
the left punch holder (use the opposite configuration for left handed persons; See Note [vii])
- To ensure the alignment of the punches,
first move the recipient punch into position and make a mark in the paraffin. And then do the same for
the donor punch. These marks should coincide precisely (See Note [viii]).
- Move the needles to the position of the first punch
with the X and Y micrometer adjustment knobs. The position of the punches over the block can be assessed
by gently pushing down on them until a mark is made in the paraffin, continuing to make adjustments with
the micrometer knobs until the desired position is attained.
- Zero the
Array Construction (assuming 0.6 mm punches)
At this point it may be necessary to adjust the depth stop, which dictates how deep the punch will be
in the recipient block. Tightening or loosening the nut at the top left of the turret does this. The
nut stops the downward movement of the turret to achieve the desired depth.
- Apply pressure to the top of the turret to
bring down the needle. A squeezing motion is used to push the tissue core into the paraffin.
- Rotate the arm of the punch to the left and then back to the right, while maintaining
pressure on the turret top. This rotating motion helps free the core from the recipient paraffin block.
- Release the pressure on the turret, allowing the springs to raise the turret to
its resting position.
- Press down on the stylus to eject the paraffin core from
the punch and examine its length.
- Make any necessary adjustment with the depth
- Swing the turret to the right to allow the donor needle to be brought
into proper position.
- Place the bridge over the recipient block.
- The donor block with the area of interest circled is placed on the bridge under the
needle and a punch is taken from the inscribed sample area by repeating the procedure for removing the
paraffin core from the recipient block (see Note [ix])
- The bridge and donor block are removed.
- The core is
inserted into the previously made hole by bringing the turret down until the lower punch surface is
directly over the hole that was just created.
- Keep the pressure on the turret
and slowly push down on the stylus, guiding the core into the hole while expelling the tissue from the
punch. This sample will have the coordinates 01, 01 from the map.
- Move the
X-axis micrometer knob to 0.8mm and repeat (see Note
- Continue across the row until the last core has been placed.
- Now use the X-axis micrometer knob to go back to position zero and move the Y-axis
knob to 0.8mm, and repeat until the block is complete.
TMA Block Sectioning
- On completion of the array, remove it from
the base plate and place into a 37 degree C oven for 15 minutes face down on a clean glass slide. This
facilitates the adherence of the cores to the walls of the punches in the recipient block.
- Remove the slide/block combination from the oven and apply gentle and even pressure.
This evens out any irregularities in the block surface and removes the bulging of the block center that
occurs during array construction (see Note 17).
- Immediately place the TMA block
onto a block of ice and wait until the complex is completely cooled before disassembly.
- Set the temperature in the water bath to 37 degrees C. Section blocks at 4-5 um, always
placing sections on the slide in the same orientation (see Note [xi]).
- Dry the sections overnight in a vertical position.
- Place slides front to back and put into a stack, wrap tightly with Parafilm, label,
and then store at -20 degrees C (See Note [xii]).
Preparation of Cell Culture Blocks as TMA Block Donors
- Grow cells using your preferred method,
depending upon the cell line.
- When cell cultures are 50-75% confluent, detach
cells and resuspend in 10% (v/v) phosphate-buffered formalin at room temperature. The time of fixation
can vary depending upon the application; we typically fix over night.
fixed cells by centrifugation at 500 x g for 10 minutes, wash once in 1X PBS and pellet again.
- Resuspend cell pellets in an equal volume of 0.8% agarose (prepared in 1X PBS) at 42
- Prefill the tapered end of a 0.6 ml microfuge tube with
agarose and let solidify.
- Transect a 1000 ul plastic pipette tip approximately 3
mm from the end of the tip using a razor blade.
- Using the transected pipette
tip, transfer the agarose/cell mixture to the microfuge tube that was previously filled with agaraose.
- While the agarose is still melted, add a wooden toothpick to the tube. This
facilitates removed of the cells when the agarose is solidified.
- After the
agarose has solidified, remove the cell plug using the embedded toothpick and section the plug in half,
generating two cell blocks.
- Process the agarose plugs into paraffin blocks
using standard tissue processing. Several plugs can be placed into a single paraffin block.
[i] ) To put more than 600 cores into a single TMA
block, it is necessary to remove the block from the base plate and rotate the block 180 degrees. This
is due to the limitation in travel along the X-axis. If the block is removed it is difficult to realign
the rows and columns.
[ii] ) During construction the block will begin to bulge
up. Incorporating space between cores minimizes this. Greater spacing will cause less bulging.
[iii] ) The use of control tissue is optional, but often
is quite helpful when examining new and existing immunohistochemical or other markers. In addition, it
can serve as a quality control during block construction. For example, once a number of tumor tissues
with similar morphologies are placed into a TMA, there is no simple way to check if the tissues were
correctly placed. If the array was designed with liver tissue to be placed in position X5,Y3, then the
expectation is that the final block will contain such tissue in the proper location. Finding a number of
such tissues in place is quite reassuring that the block was indeed correctly constructed.
[iv]) If the area within the block that is selected
contains tissue that is quite thin, it may be advantageous to select an alternate sample. Thin donor
blocks will not yield many Tissue Array slides. In general, the tissue in the donor block should be at
least 1.5 mm thick, although at times certain types of samples may not contain this much tissue. In this
case, one must decide upon the relative merits of using a TMA versus simply cutting standard blocks and
staining standard slides. For very precious specimens such as brain biopsies we have successfully
prepared TMAs using quite thin specimens.
[v]) Although other types of paraffin can be employed,
in our recipient blocks we use Paraplast X-tra, which is softer than standard paraffin. We also use deep
molds. Care should be exercised to avoid trapping air bubbles under the cassette.
[vi]) It is important to face off the recipient block
using a dedicated rotary microtome prior to use. This helps assure that the block face is smooth and
that all the arrays that are made will be in the identical plane. This minimizes the amount of block
realignment that will be necessary during sectioning and helps to optimize the number of complete
sections that an array block will yield. The backside of the block should also be checked to to ensure
that it is level as well.
[vii]) The most agility is required while expressing the
tissue into the recipient block. A margin of 2.5 mm of paraffin is recommended around the entire array.
This prevents the recipient block from cracking during construction.
[viii]) Adjustments can be made to the positions of the
punch in the holder. There are four screws holding the v-block, they all need to be loosened before
turning the front to back setscrew. Clockwise turning will bring the punch forward. Counter clockwise
will bring it backward. Left-right alignment is achieved using the setscrews on the left and right
sides of the turret.
[ix]) There is no depth stop to aid in removal of tissue
samples from the donor block. This is refined with some practice.
[x]) It is easiest to move horizontally across the map
because the X-axis adjustment knob is more accessible.
[xi] ) Also, make sure the section is placed parallel
with the slide edge. Having the sections in the proper orientation on the slide greatly facilitates
scanning of the slide with automated slide scanners.
Acknowledgements: We would like to thank Cellie Southerland for her diligence and expertise in TMA
construction . We would like to thank Mark A. Rubin and the members of his laboratory for illustrating
hands on techniques to help us get started in the construction of tissue microarrays. We also thank
Gerrun March for perseverance in scanning TMA slides. We thank James Morgan and Brian Razzaque for
continued help with the TMAJ database
[xii] ) When sectioning a TMA block, we typically cut
20 sections at a time. No published reports on the best methods of storing TMA slides are available.
While some are storing under nitrogen gas, we store unstained sections without baking the slides and at
-20 degrees C. We have had good success with this method with several antibodies over several months
time. However, the long term effects of this storage method have not been examined.
34) Several accessories have been added to the Beecher product line, that are useful and worth
mentioning. The Four-Block indexer allows four replicate blocks to be made simultaneously. The Depth
Stop Kit is used to help determine the depths of the punches in the donor blocks. Although an
experienced person may not need these a novice will find them useful. Extended hours of making tissue
micro arrays can lead to wrist fatigue and aggravate carpel tunnel syndrome. The Motorized Positioner
for the Manual Arrayer can alleviate this.
35) Cores that are placed too deeply may be removed with the smaller size needle (recipient needle).
Then, an additional core can be inserted. This process should not be considered routine for several
reasons. The hole in the recipient block may become enlarged and some residual tissue will be left
behind. Cores placed erroneously may also be removed in the fashion. Be sure to make a note whenever
this is necessary because the residual tissue may yield incorrect data.
36) The depth of the recipient core can be set by the Depth stop which is located in the upper left
hand corner of the turret. But there is no depth stop incorporated into the design of the Tissue Arrayer
that will dictate the depth of the donor core. This depth cannot be the same when you are going from one
block to another because tissue blocks are of varying thickness, which will determine the limit of the
depth of the cores that can be taken. Beecher has recently engineered a Depth Stop Kit, which will aid
in extracting reproducible lengths of donor cores.
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