This document discusses tissue microarrays (TMAs), which allow analysis of hundreds of tissue samples on a single slide. It describes how TMAs are constructed by taking small tissue cores from donor blocks and embedding them in a recipient block. The advantages of TMAs include high throughput analysis and relatively low cost. Various types of TMAs are used for applications like immunohistochemistry, in situ hybridization, and analyzing protein/DNA expression. The document outlines the steps to construct a TMA, including defining the research question, selecting cases, determining core size and number, making a map, and embedding the cores. Quality controls and limitations are also discussed.

1. TISSUE MICROARRAY
Mr.Mahmoud Ibrahim

4. 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.

5. • 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.

6. HISTORY
• 1986 : H.Battifora – “ Multi tissue tumor
block” – Sausage method
• 1998 : Kononen et al. – “ Tissue micro
array”(TMA)
• Most noteworthy development in
histopathology techniques in the last
decade

7. • Tissue microarrays
• Tiny cores of tissue arranged onto a glass
slide
• Analysis of hundreds of tissue specimens
in a single experiment

8. Characteristics of Tissue Microarrays
• 50 - 500 tissues or more can be analyzed per
slide block
• high throughput
• relatively low cost
• can be stained with a variety of stains such as
H & E
• Stained slides can be analyzed with a wide-
variety of techniques

9. • automated analysis and data collection with
many techniques

10. Applications of Tissue Microarrays
• Immunohistochemistry
• Staining of : H & E, HC, ISH,
• In situ hybridization
• Fluorescent in situ hybridization (FISH)
• In situ PCR

11. • RNA or DNA expression analysis
• TUNEL assay for apoptosis
• Morphological and Clinical
Characterization of many Patient
Tissues

12. Why use tissue microarrays
• Scanty tissue,
• either in amount or case number
• The difficulty of working with human
tissue
• scarce resources

13. Types of TMA
• First, TMAs can be categorized according
to their
A. Material of origin.
1. Tissue microarray‘
if they are constructed from paraffin
embedded material

14. • 2. Cryoarrays
• TMAs have been constructed from
frozen tissue

15. • Secondly, TMAs can be categorized
according to their application or
depends on the question being asked
within the study as:

16. • 1- Cryo - Tissue Microarrays
• 2- Multi-tumours Tissue Microarrays
• 3- Progression Tissue Microarrays
• 4- Prognosis Tissue Microarrays
• 5- Cell line arrays

17. • 6- Random tissue/tumor arrays
• 7- Consecutive case array
• 8- Tumor characteristic-based array
• 9- Outcome based arrays
• 10- Other special types

18. Cryo - Tissue Microarrays
• Cryo-TMAs uses frozen tissues which
are embedded in an optimal cutting
temperature compound.

19. • These cryo-TMAs due to their freezing
are superior to formalin fixed tissues
for RNA and protein analysis.

20. 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.

21. 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.

22. 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.

23. Random tissue/tumor arrays
• These arrays contain tissues from multiple
sites and contain tumor and or non-tumor
tissues.
• Used as quality control.
• In addition they can also be used as
discovery tools.

24. 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.

25. Outcome based arrays
• These are the most valuable and most
difficult to generate as 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.

26. • 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.

27. Team Required for TMA Construction
• Construction of TMA is a team effort.
• The first and foremost question that needs to
be answered is
• why is the TMA being constructed?
• This will decide the composition of the team.

28. • For the generation of the simplest of
TMA, a technologist might be all that is
required.
Most TMA synthesis will require close
collaboration with the pathologist

29. • t 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

30. LIMITATIONS IN USING TMAs
• Sample fixation and embedding has great
impact on the quality of TMA sections.
• buffered formalin modifies the RNA
molecule likely leading to damaging effect
on RNA by altering antigenic epitopes
structure

31. • Small size of the TMAs may not provide a
glimpse of the entire tissue profile.
• In certain heterogeneous cancers, such as
prostate adenocarcinoma and Hodgkin
lymphoma, small cores may not be
representative of the whole tumor .

32. • A tumor tissue may comprise of many
different histological areas within itself, such
as regions of apoptosis, necrosis or increased
proliferation etc and it may not be possible to
sample all areas in one tissue core

33. • Care should be taken while taking the cores
from the original tissue and focus should be
made on the purpose of investigation‘.

34. When Not to Use TMAs
• In case of there is marked heterogeneity
within tumors;
• In certain tumors such as glioblastoma
• In case of study rare or focal events
• such as number of immune cells in tumors.

35. • In case of study facets of tumor biology
• such as interactions between the tumor and
it‘s stromal
• as these stromal components may not be
adequately represented in the cores.
• The use of large cores (2 mm) has been
advocated for these types of studies

36. Steps Involved in TMA Construction
• Step 1. Define the question;
• TMAs are created to answer specific
questions.
• define this question at the outset.
• The question will help define the number of
cases and cores that need to be used in the
generation of the TMA.

37. • For example a TMA containing 20 cases
might be sufficient for routine quality
control/ assessment but is not enough for
biomarker assessment.

38. Step 2. Review the cases to be included
in the TMA
• Pull all the cases to be included in the TMA
together.
• A fresh H&E slide may be obtained to ensure
that the slide is representative of the block.
• It is useful to mark multiple areas from more
than one block,

39. 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.

40. • 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.

41. • inflict little damage on the donor and
recipient blocks and
• cores are easier to remove and replace
from these blocks.

42. • The larger core sizes :
• have the advantages 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

43. • greater chance of the blocks being
broken or cracked during the TMA
generation process.

44. 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.

45. • When using 0.6 mm sized cores, it is
typical to use a minimum of 3 cores per
case.
• Three 0.6 mm cores are still better than
one 1.0 mm core,

46. • Three 1.0 mm cores could result in
destruction of the donor
block
• Studies that have used 1 mm core
punches have tended to use two cores .

47. • 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
IHC methodology

48. • Cores should start at least 3 mm away from
the block edges, to prevent the paraffin from
cracking.
• For these reason, it is typical for most
workers to put somewhere between 100 and
300 of 0.6 mm cores in a TMA block

49. • The numbers of cores to be arrayed in
one paraffin block is selected according
to the requirements of the tests.

50. • 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.

51. • 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

52. • Controls should be placed on each TMA
block for
• quality control and
• to address tumor heterogeneity.

53. • Three types of control tissues may be
used:
• A. Tissue-specific controls
• B. Biology-associated controls
• C. Organ system controls

54. 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.

55. 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.

56. • 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.

58. 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

59. 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.

60. What are the advantages?
• There are numerous advantages to this
technology including:
• Amplification of a scarce resource
Experimental uniformity
Decreased assay volume
Does not destroy original block for diagnosis

61. • Another significant advantage is that only a
very small (a few µl) amount of reagent is
required to analyze an entire cohort.
• This advantage raises the possibility of use of
tissue microarrays in screening procedures
(for example in hybridoma screening), a
protocol that is impossible using conventional
sections.

62. • It also saves money when reagents are costly.

63. TMA Analysis
• The analysis of the TMA has 2
components.
• analysis of the slides recording of the
data.
• data analysis.

69. Arrayers
• There are several different arrayers on
the market today
• Automated arrayer
• Manual arrayer
• Portable quick ray

70. Smoothing and sectioning
• The array block must be smooth and level
before 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.

71. • 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

72. Troubleshooting and tips
• Core does not come out of the punch
easily
• suggesting that the punch tip is bent or
distorted.
• It is advised to change the punch.

73. • • Tissue core was pushed too deep.
• Advise removal of the sample with the
small punch and place a new sample in
the same position

74. • Insufficient spacing of cores.
• This can cause minor cracks or stress on the
core when sectioning.
• Loss of tissue on water bath.
• This may be due to folds, wrinkles, and
mishandling of ribbon.

75. Ref
• IHC Staining Methods, Fifth Edition, Chapter
7 Tissue Microarray —Construction and
Quality Assurance
• Rashmil Saxena BFA, HT(ASCP)CM and Sunil
Badve MD, FRCPath