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Optimizing Tissue Micro Array, Technique and Outcome Troubleshooting
By Yahya I. Elshimali, MD, Assistant Professor of Pathology and Laboratory, Medicine, David Geffen School of Medicine, UCLA Medical CBy Yahya I. Elshimali, MD, Assistant Professor of Pathology and Laboratory, Medicine, David Geffen School of Medicine, UCLA Medical Center. LA, California, and Rana M. Issa,
MD, Attending Pathologist, Department of Pathology, Hamburg
University, Germany
Tissue Micro Array (TMA) is a high throughput analysis of
paraffin embedded tissue specimen used for gene validation by
performing immunostaining and fluorescence in situ hybridization
(FISH). It was described first by Kononen et al. and published
in Nature Medicine in 1998(1).
Multiple studies were published about building, validation and
using TMA(1,2,3) and TMA became a very important tool to
research laboratories and clinical applications in centers with
a large volume of molecular testing.
Usually, a triplicate of cores from one tissue type are obtained
from donor blocks and placed in one receiver paraffin block in
designed orders of rows and columns. Analysis of the micro array
can be done in a short time, cost effectively, with high
accuracy, less labor, and it is with ease of interpretation and
minimal tissue damage(4,5).
The quality of making tissue micro array blocks is very
important since it affects the final scoring results for stained
spots on glass slides. However, a variety of difficulties and
technical problems can be faced while making TMA blocks and it
is very important to avoid such difficulties.
The following subjects will be discussed and addressed in this
article:
1. Optimizing the technique of TMA
2. Manually versus automated tissue micro array
3. Validation tissue micro array
4. How to make TMA more effective and efficient
5. How to generate more data from TMA
Optimizing the technique of TMA
1. Sources of blocks
2. Cutting paraffin blocks of tissue micro array and slide
storage
3. Increasing the number of produced TMA slides
4. Missing spots from the array
1. Source of Blocks
Most tissues used in TMA are obtained from formalin fixed
paraffin-embedded (FFPE) tissue blocks from an archive of
surgical pathology in a Facility. These blocks are made for
diagnostic purposes, not for TMA purposes, so the tissues are
not aligned to fit the purpose of tissue array.
As a result, the length of tissue in the core from the donor
block will be varied and the histology level may not be similar
in all obtained slides from the receiver block. On the other
hand, some blocks that are very important for tissue type do not
contain enough material.
To solve this concern, it is necessary to ensure having enough
blocks with sufficient material to be cored without damaging the
diagnostic value of the blocks that are kept for any legal issue
in the future.
Blocks with tiny or limited material are not ideal. Some slides
that have small biopsy, such as small cell carcinoma of the
lung, could be the only material in the block and, in such
cases, is not used unless it has extra material or
unless the case has expired and has been documented.
On bench area, and after the diagnostic blocks are made, the
pathologist may harvest some material (benign and malignant
tissue in two separate containers) that can be used strictly for
TMA purposes.
The submitted tissue must be aligned vertically in the donor
block to the best position and eventually the full thickness of
the tissue will have similar histology when cored and placed in
the received block.
2. Cutting Paraffin Blocks of Tissue Micro Array and Slide
Storage
Most receiver blocks are 4-5 mm in thickness and the tissue
thickness in the slide ranges from 0.3-0.5 micrometer. Ideally,
a few hundred slices can be obtained from one receiver block.
However, this may not be the case, as placing the TMA paraffin
block on the microtome to make array slides can cause many
voluble sections to be lost before mounting the microtone blade
to the full surface (all spots) of tissue block.
To avoid this difficulty, one of the solutions is to cut all
block completely at one time and then dip the slides (once and
quickly) in melted paraffin, keeping it at room temperature for
appropriate use.
In our experience, we compared the immunostaining of estrogen
receptors, progesterone receptors, p53 and Ki 67 in 5 newly made
slides from breast (normal and malignant spots) tissue
microarray block to 5 paraffin dipped slides that were made six
months ago and kept at room temperature, and we had very similar
analytical results, P < 0.001. Also, very similar analytical
results were obtained when we compared an ovarian tissue
microarray of papillary carcinoma for immunostaining with Fas
receptors, p53 and P 16, P < 0.001.
3. Increasing the Number of Produced TMA Slides
These blocks are different from the traditional blocks that are
now used in TMA; the thickness of these blocks is 1-1.5 cm
compared to 0.5 cm.
For the same amount of work, money and energy, 30-50% extra
slides can be harvested compared to the traditional block.
Thickness of traditional paraffin blocks is 0.4-0.5 cm and the
thickness of newly made receiver paraffin blocks cannot exceed
that thickness. Since the length of newly placed tissue cores in
receiver block are varied (0-0.5 cm), we found that the average
slides that could be obtained from such block, and with full
arrayed spots, is about 200 slides.
Indeed, the number of slides can be increased by 30-50% per
blocks, which is dependent on increasing the thickness of donor
block from 0.5 cm up to 1-1.5 cm. This can be achieved by making
donor blocks (3.5 X 2.5 X 1-1.5 cm) from tissue that has been
collected purposely for TMA, and the receiver blocks will have
the same thickness as well. By doing so, the number of produced
slides increases without extra expense, effort or time (figure
1).
4. Missing Spots from the Array
Missing
spots from the array could be due to technical difficulties or
tissue type.
1. The
hall tissue component from placed core in the donor block can be
missed or lost, and it is important to ensure the full tissue
core is placed in a suitable space. Sometimes tissue-free
paraffin cores or exceeding one core space in the donor block,
can be made intentionally, either because there is not enough
tissue to complete the triplicate of cores of the same type or
for identification purposes.
2. Missing
spots may result from small tissue component in donor block,
dependent on different levels of sectioning.
3. Missing
spots may be due to use of adhesive tape.
Adhesive
tape has been used to place on the block surface for every
single section. After making the section, the adhered
tissue-tape is placed on adhesive slide. The slide-tissue-tape
unit will be exposed to ultraviolet light for about 45-50
seconds, then the slide will be immersed in a dissolvent liquid
to remove the tape from the glass slide. During this process, a
few, some, or all spots can be lost from the surface of the
slide.
This can
be avoided by: not putting too much pressure on the tape above
either the block or the slide, not keeping the slide in the
solvent solution for more than one minute, and avoiding air
bubbles under the adhesive tape.
4. Missing
spots due to block dimensions or edge factor.Missing
spots can occur at the edge or the center of the arrayed slide.
This can be avoided by leaving at least 4-5 mm distance from
each side of the receiver block and at least 2-3 mm between
quarters, dependent on core size and number of spots per array
(Figure 2).
5. Missing
spots due to tissue type.
Spots that are predominantly composed of fatty component, such
as omentum, breast lipid, fatty tissue, etc., are easy to
dissolve or drop due to exposure to different chemicals or
solutions.
Being very selective about the spots and avoiding fatty tissue
components as much as possible can minimize this.
6. Missing spots from the side of the array may result from
inappropriate mounting of the block on the microtome, and when
the distance between the edge of paraffin block and the
row/column is less then 4 mm.
Manually Versus Automated Tissue Micro Array
Automated TMA is very productive, time effective and quicker yet
with high quality. However, it is not practical for many arrays
and manual TMA should then be adopted.
Automated TMA works better for large surfaces of same histology,
as in soft tissue tumors. The ideal block should contain more
then 1 cm diameter of solid tumor tissue (carcinomas or
sarcomas).
Automated is not preferred when the target tissue is strips,
dots or tiny spots such as benign or normal, metaplastic or
dysplastic epithelium and randomly distributed foci of tumor
cells in the section.
However, future solution for automated array for specific
histology can be achieved by using a laser assisted robotic
system that guides the arrayed needle to the targeted marked
spot on the donor block.
Validation Tissue Micro Array
Tissue microarray is constructed from different paraffin blocks
made at different times of different paraffin quality, different
fixed material and at different times of fixation. bBecause of
these differences, validation of tissue array should be done
before it is used for any study.
Validation will be achieved by immunostaining on one slide per
array and selection of the marker will depend on tissue type.
In our selection process, if the arrayed tissue has no previous
immunostaining, then 10% of the arrayed cases will be randomly
selected for staining with specific markers, in addition to the
representative slides of tissue array and the staining is done
in the same condition. Comparison (intensity and percentage of
staining for spots/slides) is then performed. The results will
be acceptable if the stain shows similarity of ≥95%. If the
arrayed cases have immunostaining before constructing the array,
it is acceptable to stain only the representative array slide
and do the comparison with the results from the path archive.
The results will be acceptable if the similarity is ≥95%.
Recommended markers for validation as per tissue type:
| Breast tumors |
Estrogen/progesterone |
| Skin tumors |
Cytokeratin |
| Lung tumor |
Cytokeratinw/FFT-1 |
| Gastrointestinal tumors |
CK 20 |
| Genitourinary tumors |
Ck 7 |
| Kidney tumor |
EMA |
| Soft tissue tumors |
Vimentin |
| Brain tumors |
S100, neural glial fibrillary acidic protein (GFAB) |
| Neuroendocrine tumors |
Gromogranin, Synaptophysin |
| Liver tumor |
Hepar 1 |
| Thyroid tumors |
Thryglobulin, TTF-1 |
How to
Make TMA More Effective and Efficient
1. Numbers of spots per array
If donor blocks contain biopsy only, it is impracticable to
array more than 120-150 cores per block. Arraying three cores
per case is feasible and valid for studying biopsy material(6).
2. The diameter of the arrayed spots is dependent on tissue type
and ranging as follows:
6 mm
1.0 mm
1.2 mm
1.5 mm
2.0 mm
3. Failure of Immunostaining
Some times the immunostain, FISH, and mRNA in situ hybridization
(ISH) fail to give expected results. It is important to
recognize the false negative and the false positive in such
cases.
To avoid misinterpretation of molecular expression, the
following steps need to be reviewed.
1. Reagent concentration, repeat stain with multiple dilutions
on full tissue section
2. Change the antigen retrieval methods, microwave, pressure
cooker, water path, etc.
3. Modify incubation time with primary antibody
4. Compare staining with monoclonal vs. polyclonal antibody
5. Home made vs. commercially available antibody may be used
6. Different results may be obtained from different antibody
resources (mouse, goat and rabbit antibody)
7. Observe for expired or old reagent or antibody
8. Different antibodies from different producers
9. Observe for false positive due to secondary antibodies
How to Generate More Data from TMA
Obtaining more data from TMA depends on the method of building
up the array and ease of information and statistic retrieval
from scoring spots. Different arrangements for histology types
can be designed.
The following are examples for such histology, Figure 3.
Other design is randomly distributing the malignant and benign
tissue in triplicate multiplications of cores as in figure 4.
Scoring/Reading TMA spots should be evaluated and some times the
accidental finding could be more important than the study
target. Scoring should be addressed for cellular component, type
of cells and other findings at the same spot (Table 1).
References
1. Kononen, J., Bubendorf, L., Kallioniemi, A., Barlund, M.,
Schraml, P., Leighton,
S., Torhorst, J., Mihatsch, M. J., Sauter, G., and Kallioniemi,
O. P. Tissue micro arrays for high-throughput molecular
profiling of tumor specimens. Nat Med, 4:844-847,1998.
2. Validation of a tissue microarray to study differential
protein expression in inflammatory and non-inflammatory breast
cancer. Van den Eynden GG, Van der Auwera I, Van Laere S,
Colpaert CG, van Dam P, Merajver S, Kleer CG, Harris AL, Van
Marck EA, Dirix LY, Vermeulen PB. Breast Cancer Res Treat. 2004
May;85(1):13-22.
3. Molecular classification of breast carcinomas using tissue
microarrays. Callagy G, Cattaneo E, Daigo Y, Happerfield L,
Bobrow LG, Pharoah PD, Caldas C. Diagn Mol Pathol. 2003 Mar;
12(1):27-34.
4. Camp, R. L., Charette, L. A., and Rimm, D. L. Validation of
tissue microarray technology in breast carcinoma. Lab Invest,
80: 1943-1949, 2000.
5. Milanes-Yearsley M, Hammond ME, Pajak TF, Cooper JS, Chang C,
Griffin T, Nelson D, Laramore G, Pilepich M. Tissue micro-array:
a cost and time-effective method for correlative studies by
regional and national cancer study groups. Mod Pathol. 2002
Dec;15(12):1366-73.
6. Gulmann C, Butler D, Kay E, Grace A, Leader M. Biopsy of a
biopsy: validation of immunoprofiling in gastric cancer biopsy
tissue microarrays. Histopathology. 2003 Jan;
42 (1):70-6.
Rana M. Issa, MD is an Attending Pathologist with the Department of Pathology at Hamburg University, Germany.
Yahya Elshimali, MD is as an Assistant Professor of Pathology and Laboratory, Medicine at David Geffen School of Medicine, UCLA Medical Center. LA California. For article feed back contact Yahya Elishmali at Elshimali@gmail.com
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