Tissue Microarrays

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Tissue Microarrays
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What are tissue microarrays?   Tissue
microarrays are produced by a method of
re-locating tissue from conventional histologic
paraffin blocks such that tissue from multiple
patients or blocks can be seen on the same slide.
This is done by using a needle to biopsy a
standard histologic sections and placing the core
into an array on a recipient paraffin block. This
technique, originally described by in 1987 by
Wan, Fortuna and Furmanski in Journal of
Immunological Methods. They published a
modification of Battifora's "sausage" block
technique whereby tissue cores were placed in
specific spatially fixed positions in a block.
The technique was popularized by Kononen and
colleagues in the laboratory of Ollie Kallioneimi
after a publication in Nature Medicine in 1998.
Up to 600 specimens
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Hodgkin lymphoma tissue microarray construction
and evaluation hematoxylin and eosin stains
Alexandar Tzankova, Philip Went, Annette Zimpfer
and Stephan Dirnhofer, Tissue microarray
technology principles, pitfalls and
perspectiveslessons learned from hematological
malignancies. Experimental Gerontology 40
737-744 (2005)
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Figure 2 shows an overview of a completed colon
cancer array and higher magnification views of
spots from this array and a melanoma array.
Stains shown in these examples include
hematoxylin and eosin and DAB based-immunoperoxida
se. The current Beecher Instruments arraying
device is designed to produce sample circular
spots that are 0.6mm in diameter at a spacing of
0.7-0.8mm. The surface area of each sample is
0.282 mm2, or in pathologists terms, about the
size of 2-3 high power fields. The number of
spots on a single slide is variable depending on
the array design, the current comfortable maximum
with the 0.6 mm needle is about 600 spots per
standard glass microscope slide. New technologies
are under development that may allow as many as
2000 or more sections per slide. Using this
method, an entire cohort of cases can be analyzed
by staining just one or two master array slides,
instead of staining hundreds of conventional
slides, yet each spot on the array is similar to
a conventional slide in that complete demographic
and outcome information is maintained for each
case so that rigorous statistical analysis can be
done as rapidly as the arrays are analyzed.
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What are the advantages?   Advantages to this
technology Amplification of a scarce
resourceExperimental uniformity Decreased assay
volumeDoes not destroy original block for
diagnosis
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Coordinates of the tissue microarray (left) and a
panoramic view of the corresponding non-Hodgkin
lymphoma tissue microarray slide (right)
hematoxylin and eosin stain
Alexandar Tzankova, Philip Went, Annette Zimpfer
and Stephan Dirnhofer, Tissue microarray
technology principles, pitfalls and
perspectiveslessons learned from hematological
malignancies. Experimental Gerontology 40
737-744 (2005)
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Are the small histo-spots representative of a
whole section? The major potential limitation
of this technique is tissue volume. Skeptics
claim that the amount of material analyzed is too
small and potentially not representative of the
entire tumor. In a reproducibility study, the
Sauter group found identical Kaplan Meier curves
were generated by analysis of 4 unique sets of
spots from the same patients (Torhorst et al, AJP
2001). Thus although any given histo-spot may be
negative on a given array, the statistical power
of analysis of hundreds or thousands of cases
eliminates the affect of variability of a single
data point in the ultimate conclusions. Our own
study to assess the number of histo-spots
required to obtain an equivalent result to a
tissue section using the standard breast cancer
prognostic markers (Estrogen and progesterone
receptors and HER2 oncogene) shows that analysis
of only 2 histo-spots results in gt95 accuracy
(see figure in Camp et al, Lab Invest, 2000).
More recently, numerous other studies have found
similar results.
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Minimal influence of the number of evaluated
tissue microarray cores on the final results as
exemplified by diffuse large B-cell lymphomas. Of
note, the proportion of positive cases for
consistently expressed markers such as CD20 does
not change by increasing the number of analyzed
cores, while that of BCL2 and BCL6 (each
expressed only in a proportion of malignant
cells) slightly increases. Even if the results
for all three markers are combined, the Final
outcome increase is of marginal significance when
the findings from the second and third cores are
added. p-computed by the paired sample t-test.
Alexandar Tzankova, Philip Went, Annette Zimpfer
and Stephan Dirnhofer, Tissue microarray
technology principles, pitfalls and
perspectiveslessons learned from hematological
malignancies. Experimental Gerontology 40
737-744 (2005)
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Hodgkin lymphoma tissue microarray core stained
for p53 left overview, right detailed view of
the framed region immunoperoxidase stain.
Alexandar Tzankova, Philip Went, Annette Zimpfer
and Stephan Dirnhofer, Tissue microarray
technology principles, pitfalls and
perspectiveslessons learned from hematological
malignancies. Experimental Gerontology 40
737-744 (2005)
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Easily identifiable follicular lymphoma on a
tissue microarray core of only 0.6 mm diameter
hematoxylin and eosin stain (left), anti-BCL6
immunoperoxidase stain (right).
Alexandar Tzankova, Philip Went, Annette Zimpfer
and Stephan Dirnhofer, Tissue microarray
technology principles, pitfalls and
perspectiveslessons learned from hematological
malignancies. Experimental Gerontology 40
737-744 (2005)
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Figure 1. Immunofluorescent Images Used in
Automated Quantitative Analysis of Tissue
MicroarraysThe antibodies used for
immunofluorescence were rabbit pan-cytokeratin
antibody from DAKO (Glostrup, Denmark) Estrogen
Receptor antibody (mAb clone 1D5, DAKO) and DAPI,
allowing for differential fluorescent tagging of
each. A. Cytokeratin staining (Cy2, green) of the
breast cancer TMA core shows strong staining of
epithelial tissue, which is used to define a
binary mask for the tumor region to separate it
from the surrounding stroma. B. Top right DAPI
(blue) stains all nuclei in the specimen within
both tumor and stromal regions. This is used to
define the subcellular compartment of 'nuclei'.
C. Estrogen Receptor (ER) staining (Cy5, red)
shows nuclear staining. Cy5 is used as for the
staining of the target of interest since it is
outside the auto-fluorescence spectrum of tissue.
D. This three-color overlay image illustrates the
separation of epithelial tumor (green regions)
from the stroma, which stained only with DAPI.
The overlay of the ER staining onto the
cytokeratin and DAPI images shows that ER stains
nuclei only within the breast tumor region and
not the stromal nuclei, resulting in a magenta
color.

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Expression of BCL2 in diffuse large B-cell
lymphomas (left) immunoperoxidase stain.
Clustering of protein expression in diffuse large
B-cell lymphomas two distinct clusters defined
by the co-expression of CD10 and BCL6 or
expression of MUM1 and CD44 s, respectively, can
be identified cumulative data obtained on
consecutive TMA sections greennegative cases,
redpositive cases (right). Note that BCL2 is
almost exclusively expressed in CD10-negative and
MUM1-positive cases
Alexandar Tzankova, Philip Went, Annette Zimpfer
and Stephan Dirnhofer, Tissue microarray
technology principles, pitfalls and
perspectiveslessons learned from hematological
malignancies. Experimental Gerontology 40
737-744 (2005)