Tissue Mircoarray

Moderator : Dr . Netra S
Layout

• Introduction
• History
• Characteristics of TMA
• Types of TMA
• Team required for TMA construction
• Steps Involved in TMA construction
• TMA Analysis
• Applications of Tissue Microarrays
• Advantages of TMA
• Limitations in Using TMAs
• Recent advances
• References
 Introduction
• A microarray is an orderly
arrangement of samples onto a matrix.

• Method of relocating tissue from

conventional histologic paraffin wax
blocks/frozen sections such that tissue
from multiple patients can be seen
on the same slide.
• This is done by using a needle to biopsy
a

standard histologic tissues and

placing the core into an array on a
recipient paraffin block.
 HISTO
RY– first described “ Multi tissue
• 1986 : H.Battifora
tumor block” .

• 1998 : Kononen et al. – first developed “ Tissue

micro array”(TMA) technique.

• Most noteworthy development in histopathology

techniques in the last decade.
 Characteristics of Tissue Microarrays
• 50 - 500 tissues or more can be analyzed per
slide block
• can be stained with a variety of stains such as H
&E
• automated analysis and data collection with
many techniques
 Types of TMA
• TMAs can be categorized according to their

A. Material of origin.

B. according to their application

A. Material of origin.

I.Tissue microarrays -if they are constructed

from paraffin embedded material. This technique
offers fine morphological details but can
compromise antigenicity.

II.Resin Tissue microarrays- If the tissues are

arrayed into a resin recipient block like glycol
methacrylate resin. Resin tissue micro arrays
provides enhanced tissue morphology along with
good antigenicity and thin section preparation.
III. Cryoaarays- if they are constructed from
frozen tissue. This newer technique was
developed to overcome difficulty associated with
antigenic alteration and mRNA degradation in
case of FFPE tissues. Frozen tissues embedded
in OCT compounds proved to give enhanced
results with DNA, RNA and protein analyses.

IV. TMAs have also been constructed from

paraffin embedded cell lines or cell blocks.
2. According to their application

• Multi-tumours Tissue Microarrays

• Progression Tissue Microarrays
• Prognosis Tissue Microarrays
• Cell line arrays
• Random tissue/tumor arrays
• Consecutive case array
• Tumor characteristic-based array
• Outcome based arrays
Multi-tumours Tissue Microarrays

• Multi-tumour TMAs have many

different types of tissues aligned on the
slide.
• The technique was first used by Kononen et
al.,1998.
 Progression Tissue Microarrays

• This type examines different stages of

tumour (or disease) progression within a
given organ.
• For example, examination of tumours in
the breast.
 Prognosis Tissue Microarrays
• Disease samples such as tumour biopsies can be
taken from patients and examined.
• These samples can be used for clinical follow-
ups to monitor the patient's progression.
• Data is then analyzed and compared with other
clinical data.
Expression of Extracellular Matrix Metalloproteases Inducer on BM
Micrometastatic and Primary Mammary Carcinoma Cells
 Random tissue/tumor arrays

• These arrays contain tissues from multiple

sites and contain tumor and or non-tumor
tissues.
• Used as quality control.
 Cell line arrays

• These arrays consist of normal or cancer cell

lines that are grown in culture.
• The major function of these arrays is survey
the presence of proteins that are known to be
present in one or more the cell lines.
 Outcome based arrays

• They involve collation of tissues from

patients that have the same disease and
have been more or less similarly treated
and followed up for a significant
period of time.
• The period of follow-up depends on the type of
disease or tumor being studied.
• These types of arrays are mostly used to evaluate
prognostic or predictive biomarkers.
 Team Required for TMA Construction

• Construction of TMA is a team effort.

• a technologist

• close collaboration with the pathologist

• It is a good idea to involve biostatisticians from

the onset rather than just asking them to do the
data analysis.
• Outcome based TMAs may need input from or
participation of treating physicians/oncologists
TMA Construction and Generation
of Grid
 Steps Involved in TMA Construction
• Step 1. Define the question;
• The question will help define the
number of cases and cores that need to
be used in the generation of the TMA.

• For example a TMA containing 20 cases

might be sufficient for routine
quality control/ assessment but is not
enough for biomarker assessment.
Step 2. Review the cases to be
included in the TMA

• All the cases to be included in the TMA are

reviwed.
• A fresh H&E slide may be obtained to ensure
that the slide is representative of the block.
• Area of interest is marked using markers.
 Step 3. TMA core size and number of
cores
Size of the cores:

Typical core sizes used for TMA constructs are

0.6 mm,1 mm, 1.5 mm and 2 mm.

Many workers consider the small 0.6 mm cores as

the standard of practice.
• smaller core diameters :

• allows for a greater number of cores to be

extracted from the lesion

• Allow for a greater number of cores that can

fit into the TMA block.
• inflict little damage on the donor and
recipient blocks and
• cores are easier to remove and replace from
these blocks.
• The larger core sizes :

• have the advantage of being more robust

• the cores are more difficult to damage during
handling.
• can lead to increased likelihood of difficulty
in extracting the cores from the blocks with
greater chance of being broken or cracked
during the generation process.
 Number of cores

The optimal number of cores, to be included

in the TMA, is

• marker dependent

• can vary depending on the degree of

tumor heterogeneity.
• 0.6 mm sized cores uses a minimum of 3 cores per
case.
• Studies that have used 1 mm core punches have
tended to use two cores
• Three 1.0 mm cores could result in destruction
of the donor block
• Therefore, three 0.6 mm cores are still better
than one 1.0 mm core
Density:

• It is the maximum number of cores that should be

placed on a single block

•It vary depending on core size,

block size and the study to be

conducted.
• Cores should start at least 3 mm away from the
block edges, to prevent the paraffin from
cracking.
• Therefore , it is better to put the core between 100
and 300 of 0.6 mm cores in a TMA block
• Distance:
• The distance between cores should NOT
exceed the core diameter.
• It is easier for the microscopist to follow the
rows and columns if he/she can ―lead from one
core to another.
• If the distance between cores is large, it difficult
to follow the chain of cores and may result in
skipping of lanes and false recording of data
when performing manual interpretation

• Controls should be placed on each TMA block

for quality control and to address tumor
heterogeneity
Step 5. Make a TMA map depicting
the layout
• This will serve as a guideline to in order
arrange blocks and sequence in which they
need to be arrayed.
• Thus the TMA map will contain the exact
location of each case, including the duplicate
samples, and controls are located.
 Step 6. Creating the TMA itself
• Instrumentation:
• For TMAs to be made from valuable cases
with scant materials, it is necessary to use
instruments.
• The simplest of these consist of hand-held
punches and are generally not very useful for a
serious TMA project, where it is necessary to use
at least an intermediate grade device.
• Fully automated devices additionally have
integrated computers that can be programmed
to select the donor sites from different blocks
and transfer them in the recipient block.
 Donor block
• The thicker the donor blocks the more
the number of useful sections can
obtained from the TMA.
• Core punches should be pushed gently into
the TMA block, and not too deeply as this
can damage the needle as well as the block
 Recipient block
• It is best to place the cores towards the center of
this block in order to prevent cracking of the
block.
• After the cores are inserted, place the TMA in
37 °C overnight, and then on the cold plate of
the tissue embedding station with subsequent two
to three 1-hour cycles of hot/cold to temper the
array.
 Smoothing and sectioning
• The easiest way to do this is to heat a clean

microscopic slide to around 70–80°C and

touch it to the array block surface.
• The surface of the block will begin to melt.

• Move the slide in a circular motion and place

the slide and block in the refrigerator or
freezer
 TMA Analysis

The analysis of the TMA has 2 components.

• analysis of the slides

Manual /
automated
• data analysis.
methods
Applications of Tissue Microarrays

I.TMA technology allows parallel conventional

and molecular profiling thereby enabling
morphology, DNA, RNA and protein targets to
be analysed on multiple tissue samples.

II.TMA can be utilized for carrying out large-

scale analyses using immunohistochemistry
(IHC) and insitu hybridization (ISH)
III. TMAs significantly aids in rendition of
molecular alteration into clinical upshot.

IV. Helps in identification of new diagnostic and

prognostic disease markers especially cancer
researches.

V. TMAs can be used to associate molecular

alterations with the specific stage of tumor
progression.

VI. TMAs assist in testing and standardization of

potential therapeutic targets.
VII. TMAs have also been utilized in Xenograft
tumor assays .

VIII. Facilitates retrospective and prospective

tissue researches and cell line cytospin cell block
studies.

IX. TMAs prove to facilitate IHC staining

procedure and interpretation of external and
internal quality control. TMAs surmount
intralaboratory and interlaboratory variation
owing to differences in antigen retrieval, staining
protocols, antibodies used and in interpretation
of results.
 
X. Originally TMA technology was used in
cancer researches but, presently its scope is
expanding and reported in non-neoplastic
pathologies such as neurodegenerative diseases,
dermatological, cardiac and placental diseases
are also beginning to emerge.
 Advantages of TMA

1. Amplification of the resource:

• Ordinarily from a standard 5 mm thick
section of tissue, we can get maximum 100
sections for study.
• Whereas depending on the size of the tissue
in the original block, at least 200 core
biopsies can be taken to make TMA block.
After the construction of the TMA, we can cut at
least 1000 sections of 3 μ thickness from the
5 mm thick array block

2. . Uniformity in staining conditions:

• At the time of conventional staining, the
different tissue sections are stained at different
time (such as 10 batches of 20 slides each),
and
• the slide-to slide variation may occur due to
several variables such as antigen retrieval,
concentration of different reagents,
incubation period, washing time, etc.

• However in case of TMA, each tissue

section consists of 100–1000 core biopsies
from the different patients, and the single
section is stained that avoids all the slide-to
slide variability.
3. Faster, cheaper and reduction of
manpower:
• In TMA a single slide requires less reagents,
labour and time. Therefore, TMA saves cost,
time and total work force.

4. Original block can be preserved:

• Only a few core biopsies from the original
block are sufficient to make TMA. The
original block can be preserved and can be
used for further sectioning.
5. Effective in quality assurance program:
• Due to significant amplification of the
laboratory material, TMA can be used for
external and internal quality control program.
The TMA section can also be used for
standardization of reagents for positive
control.

6. The whole analysis of TMA is now automated

and computer can keep track of the huge data.
 Limitations of TMAs

1. Tissue heterogeneity:
This is one of the main concerns of TMA. The
tumor may vary from area to areas such as
Hodgkin lymphoma which may have different
morphologies in different areas. Therefore small
2 mm tissue may not represent the whole tumor
and finding may vary. However, several studies
have shown that TMA and whole tissue data are
almost similar
2. Less cost-effective in small series of cases:
TMA is not very effective if it is done once in a
while in a small series of cases.

3. Prone to loss the tissue:

The core biopsy tissues may be lost due to its
tiny size. Tissue rich in keratin, bone or
cartilages are likely to be lost.
4. Disorientation of the core biopsy tissue:
Due to large number of core biopsies, there is a
chance of disorientation when TMA is done
manually. Rows of empty core tissues may help
in the immediate orientation of the tissue.
Recent advances
References

1. Skacel M, Skilton B, Pettay JD, Tubbs RR. Tissue

microarrays: a powerful tool for high-throughput
analysis of clinical specimens: a review of the method
with validation data. Appl Immunohistochem Mol
Morphol. 2002 Mar;10(1):1–6.

2.  Williamson M, Naaby-Hansen S, Masters JR. 21st

century molecular biology in urology. BJU
Int. 2001;88:451–57. 

3. Christina J Kim, MD, Douglas S Reintgen, MD,

Timothy J Yeatman., MD The Promise of Microarray
Technology in Melanoma Care. Cancer
Control. 2002;9(1):49–53. 

4.Shergill IS, Shergill NK, Arya M, Patel HR. Tissue

microarrays: a current medical research tool. Curr Med
Res Opin. 2004 May;20(5):707–12. 

5.Kumar B, De Silva M, Venter DJ, Armes JE. Tissue

microarrays: a practical guide. Pathology. 2004
Aug;36(4):295–300. 
Thank you