This document discusses the process of tissue processing for histological examination. It involves several key steps: specimen identification and labelling, gross examination, fixation, dehydration, clearing, infiltration, embedding, sectioning, and staining. Fixation using formalin or other fixatives preserves tissue structure. Dehydration removes water and replaces it with alcohol or other solvents to allow for infiltration of paraffin wax. The wax infiltrates and impregnates the tissue, allowing it to be sectioned thinly for microscopic examination after staining. The document provides details on common fixation and processing methods and their purposes in preparing tissue for histological analysis.

1. Tissue processing
incorporating
Microarray
Presented by:
Dr. Surbhi Mahajan
(2nd year PG, Pathology)

2. DEFINITION
 HISTOTECHNIQUES: The techniques for processing the tissues, whether biopsies, larger specimen
removed at surgery, or tissues from autopsy so as to enable the pathologist to study them under the
microscope
 Tissue for study can be obtained from:
Biopsies
Autopsies
Surgery

3. STEPS FOLLOWED IN HISTOTECHNIQUES
Receipt & Identification
Labelling of the specimen with numbering
Grossing
Fixation
Dehydration
• TISSUE PROCESSINGClearing
Impregnation
Embedding
Section cutting
Staining
Mounting

4. Specimen identification and labelling:
1) Specimen/tissue taken from the body as biopsy by the
surgeon is put in a container containg 10% formalin and
along with a requisition form giving
• patients details like name, age, gender, MRD number
• identity of the tissue
• provisional diagnosis and other investigation findings
sent to the Pathology department for Histopathological
examination.

5. 2) In Histopathological room,
specimens are accessioned by giving them a number eg. [18 G 292]
that will identify each specimen for each patient and is registered in a
record book
3) After registration, sample is transferred to the Gross examination table

6. Gross Examination
The histopathologist examines the specimen
giving description including:
• Type of specimen
• Weight & measurement (approx)
• Consistency
• Cut section
• Mentioning any abnormalities

7. and takes tissue blocks of 2-3 millimeters thickness for tissue processing
 Tissue cassettes are generally made of plastic (Acetyl polymer)
 Standard blocks allow a sample of about 20 × 20 × 3mm to be
contained and fixed
 Tiny biopsies or small specimen can be wrapped in a filter paper and
then put in a cassette & fixed.

8. Tissue Processing
 The tissue must undergo preparatory treatment before being sectioned, entailing
to impregnation of specimen with an embedding medium to provide support and
suitable consistency for microtomy. This process is defined as tissue processing.

9. AIM :
Tissue processing is designed to remove all extractable water from the tissue,
replacing it with a support medium that provides sufficient rigidity to enable
sectioning of the tissue without parenchymal damage or distortion.
PRINCIPLE:
Tissue processing occurs due to diffusion of various substances/fluids into and
out of stabilised porous tissue. The diffusion process results from the
thermodynamic tendency of processing reagents to equalize concentrations
inside and outside the bits of tissue, which is in accordance to FICK’S LAW.

10. Factors affecting rate of processing:
1. AGITATION: increases the flow of fresh solutions around the tissue.
Automated processors incorporate vertical or rotary oscillation, or pressurized
removal and replacement of fluids at timed intervals as the mechanism for
agitation. Efficient agitation reduce the overall processing time by 30%.
2. HEAT: limited to 45degree
3. VISCOSITY: The higher the viscosity of fluid, the slower the rate of
penetration. Paraffin has low viscosity in fluid(melted) state,
enhancing the rapidity of impregnation
4. VACUUM: Impregnation time for dense , fatty tissue can be greatly
reduced

11. FIXATION
DEFINITION:
is the process by which the constituent of the cells are fixed in a physical and
partly chemical state so that they will withstand subsequent treatment with
various reagents with minimal loss of significant distortion or decomposition
and keep tissue in as life like manner as possible
AIM:
1. To prevent bacterial degeneration, autolysis & putrefaction by inactivating the lysosomal
enzymes
2. Should not distort the cellular constituents by swelling or shrinkage ad maintain close
resemblance as possible to the natural structure of tissue components

12. 3. Allow the tissue to withstand the chemicals used at various stages of
processing
4. Increase tissue consistency to permit the cutting of thin slices of tissue at
varying microns
5. Allow clear staining of the sections and increase optical differentiation of
cellular structure
6. To make the tissue useful for various special procedures like IHC etc.

13. Qualities of a good fixative:
1. Good tissue penetration
2. Prevents fixation artifacts
3. Prevents structure deformation, maintaining shape and volume (is
isotonic)
4. Preserves cellular constituents
5. Permits the recovery of macromolecules (proteins,mRNA,DNA) without
extensive biochemical modifications.
6. Safe to handle-nontoxic and nonallergic

14.  Reproducibility over time of the microscopic appearances of tissues
after H&E staining is the prime requirement of the fixatives used for
diagnostic pathology
 The fixative should be 10 – 20 times more in volume than the specimen
 No fixative will penetrate a piece of tissue thicker than 1 cm
 To date, a universal or ideal fixative has not been identified
 Fixatives are therefore selected based on their ability to produce a
final product needed to demonstrate a specific feature of a specific
tissue.

15. TYPES OF FIXATION (Based on mode of action)
1. PHYSICAL FIXATION
• Heat fixation – Simplest form of fixation; Usual mode of preparing bacteriological smears
• Microwave fixation – Speeds fixation ; Reduces time of fixation from >12 hours to < 20 minutes.
Allows rapid fixation of routine surgical specimen and esp. for processing urgent cardiac and renal
biopsies
• Freeze drying & Freeze substitution – Gives better preservation of antigenicity by either ICC or IHC.
Restricted to research work
2. Chemical fixation
• Utilizes organic or non-organic solutions to maintain adequate morphological preservation

16. Coagulant Fixatives
• Ethanol
• Methanol
• Acetone
• Acetic acid
• Picric acid
• Tetrachloro acetic acid
Cross linking fixatives
• Formaldehyde
• Glutaraldehyde
• Mercuric chloride
• Osmium tetroxide
• Chloral hydrate
Oxidizing agents
• Osmium tetroxide
• Chromate containing
fixatives
TYPES OF CHEMICAL FIXATIVES

17. • Both organic and non-organic solutions may coagulate proteins, making them
insoluble.
• Cellular architecture is maintained primarily by lipoproteins and by fibrous
proteins such as collagen;
• Coagulating such proteins maintains tissue histomorphology at the light
microscopic level.
• Are not useful in ultrastructural analysis.
CROSS-LINKING FIXATIVES:
Are selected secondary to their potential actions of forming cross-links within
and between proteins and nucleic acids
COAGULANT FIXATIVES:

18. Based on components present
Simple fixatives
Formaldehyde
Glutaraldehyde
Ethyl alcohol
Compound fixatives
Formalin based
Mercurial fixatives
Dichromate fixatives
Picric acid fixatives
Alcohol containing
fixatives
10%NBF, 10%NB formol saline
Zenker’s solution, Helly’s solution
Regaud’s, Moller’s
Bouin’s solution
Carnoy’s and acetic alcohol formalin

19. FORMALDEHYDE FIXATION
 Formaldehyde in its 10% neutral buffered form (NBF) is the most common
fixative used in diagnostic pathology.
 Pure formaldehyde is a vapor that, when completely dissolved in water forms
a solution containing 37-40% formaldehyde; this aqueous solution is known
as ‘formalin’ and is commercially available as 40% w/v solution and is taken as
100% formalin

20. 10% formalin contains (v/v)
1. Formalin (40% formaldehyde dissolved in water): 10mL
2. Water: 90mL
10% Neutral Buffered formalin –
 Tap water = 900 ml
 Formalin (37% formaldehyde solution) = 100 ml
 Sodium phosphate, monobasic, monohydrate = 4 g
 Sodium phosphate, dibasic, anhydrous = 6.5 g
 pH should be 7.2-7.4
It prevents the formation of formalin pigment

21. PRINCIPLE:
In the first step, aldehyde groups in formalin form complexes by forming
links (methylene bridges) between protein molecules
These methylene bridges subsequently react with several side chains of
protein to form reactive hydroxymethyl side groups
This cross-linkage does not harm the structure of proteins greatly, so that
the antigenicity is not lost
However, thus cross-linking is reversible by simple washing in water

22.  Washing for 24 hours removes about half of reactive groups, and 4 weeks
of washing removes up to 90%
Fixation time and temperature:
 An average tissue requires 10-20 times its volume of formalin/NBF for
adequate fixation and immersion for 24hrs at room temperature
 If volume of tissue is small, a minimum period of 8-12hrs is used
 If temperature is raised to 45degree Celsius, fixation time is shortened

23. When formaldehyde dissolves in
an unbuffered aqueous solution,
it forms an acid solution – Acid
Formalin(pH 5-5.5),
which decreases the quality of
nuclear staining and
leaches out hemosiderin resulting
in formation of brown black
pigment also known as acid
hematin.

24. Removal of formalin pigment before staining:
Schridde’s method:
Verocay’s method: Treat sections for 10min with a mixture of 100mL
of 80% alcohol and1mL of aqueous potassium hydroxide followed by
thorough washing in water
Kardasewitsch’s method:
Lillie’s method:
Picric acid method: Treat the sections in saturated solution
of picric acid for 5minutes to 2hours

25. ADVANTAGES :
1. Good penetration and fixation
2. Easy availability & cheap
3. Does not cause excessive tissue hardening
4. Preservation of fats, myelin, nerve fibres, amyloid and hemosiderin
DISADVANTAGES:
1. Irritant to the nose,the eyes and mucous membranes
2. On storage it becomes cloudy due to formation of paraformaldehyde,
which can be prevented by adding 11-16 % methanol in commercial
aldehyde
3. Formation of brown black formalin pigment ,acid hematin.
4. Unsuitable for demonstration of of fats and enzymes
5. It has a denaturing effect on proteins, hence unsuitable for fats and
electron microscopy

26. CHARACTERISTICS OF OTHER FIXATIVES
GLUTARALDEHYDE: Dialdehyde. The cross-linking is irreversible.
Extensive cross-linking by glutaraldehyde results in better preservation of
ultra structure. Used for electron microscopy
Osmium tetroxide: best characterized by its reaction with unsaturated
bonds within lipids and phospholipids. Used after postfixation with
glutaraldehyde in electron microscopy to retain lipids.

27. Zenker’s solution: good fixative for bloody(congested) tissues,
reticuloendothelial tissues including lymoh nodes,spleen, thymus and
bone marrow..They are also good for trichrome stains
Bouin’s solution: is an excellent general fixative for connective tissue
stains. The yellow color can be removed with 70% ethanol, lithium
carbonate, or another acid dye, separately or during the staining sequence.

28. Renal biopsies
Renal core biopsies should be subdivided into three and each piece
should contain adequate numbers of glomeruli
Each portion is then preserved, depending upon the method to be used
for analysis:
• NBF for routine histology
• Buffered glutaraldehyde (pH 7.3) for ultrastructural analysis
• Snap frozen in isopentane and liquid nitrogen for immunofluorescence
examination.

29. Factors affecting quality of fixation
1.Buffer and pH :physiological range, pH 6-8. If allowed to fall to a lower
pH this can produce formalin pigments. Common buffers include phosphates,
bicarbonates
2.Duration of fixation and Size of tissue
The depth (d) reached by a fixative is directly proportional to the square root
of duration of fixation (t) and expressed this relation as
d = k √t (k=Constant of diffusibility).
Gross specimens should not rest on the bottom of a container of fixative:
they should be separated from the bottom by wadded fixative-soaked paper
or cloth, so allowing penetration of fixative or processing fluids from all
directions

30. 3.Temperature of fixative: microwaves therefore have been used to
speed formaldehyde fixation by both increasing the temperature and
molecular movements
4.Concentration of fixative: concentrations of formalin above 10%
tend to cause increased hardening and shrinkage. Ethanol concentrations
below 70% do not remove free water from tissues efficiently
5.Osmolality :Hypertonic solutions give rise to cell shrinkage whereas
hypotonic and isotonic fixatives result in cell swelling and poor fixation.
With electron microscopy, the best results are obtained using slightly
hypertonic solutions (isotonic solutions being 340 mOsm) adjusted using
sucrose.

31. Post-fixation treatment:
Special fixation techniques may require additional steps before processing is
initiated like
Picric acid fixatives (Bouin’s) form water-soluble picrates making it necessary
to place the tissue cassettes directly into 70% alcohol for processing
Treating mercury fixative containing sections with iodine mixture (Lugol’s
Iodine + Sodium thiosulphate)

32. Improper Fixation: delay in fixation or inadequate
a. Altered staining quality of cells
b. Cells appear shrunken and show cytoplasmic clumping
c. Indistinct nuclear chromatin with nucleoli sometimes not seen
d. Vascular structures, nerves and glands exhibit loss of detail
e. Impression of scar formation or loss of cellularity.
AUTOLYSIS DUE TO
IMPROPER FIXATION

33. DECALCIFICATION
 It is the process of removal of inorganic calcium salts from the tissue
to make it amenable for sectioning
 The tissue must be fixed adequately before decalcification as acid
solutions used in decalcification are injurious to the organic ground
substance of the bone
 Selected bone is placed in a 10%NBF for 24-48hrs

34. The various methods used for decalcifying are
Acid decalcification: Inorganic acids- Nitric acid
Hydrochloric acid
Organic acids- Formic acid
Acetic acid
Picric acid
Chelating agents- Ethylenediaminetetraacetic acid
Ion exchange resins
Electric ionization
Surface decalcification Needed when partially decalcified bone/unsuspected
mineral deposits in soft tissue are found during paraffin
sectioning. the exposed surface in a paraffin block is
placed face side down in 5% HCL for 1hour

35. End point decalcification
• Probing the tissue with the needle
• Chemical tests
• Bubble test
• Radiography
Neutralization of acids
Chemical neutralization is accomplished by immersing decalcified
bone into either
• Saturated Lithium Carbonate Solution or
• 5-10% Aqueous Sodium Bicarbonate Solution for several hours.

36. DEHYDRATION
It is the process in which the water content in the tissue to be
processed is completely reduced by passing the tissue through
increasing concentrations of dehydrating agents.
AIM: Dehydration is done so that the wax i.e Paraffin wax, which is
used for impregnation, can be easily miscible as it is immiscible with
water.

37. PRINCIPLE:
Water is present in tissues in free and bound (molecular) forms.
Being hydrophilic, these dehydrating agents act by attracting water
molecules from the tissue or
affect dehydration by repeated dilution of aqueous tissue fluids.
 Tissue is passed through series of progressively more concentrated
alcohol baths
 Best accomplished by use of graded alcohol like 70-95%

38.  Transfer of tissue directly to higher conc. is risky since it is liable to
cause tissue shrinkage
 The concentration of the first fixative depends on the the fixative and
size and type of tissue
 Delicate tissue such as embryo and brain needs lower alcohol conc
(50% to start with) and smaller intervals between two stengths of
alcohol
 Tissue immersed in alcoholic fixatives such as Carnoy’s fluid may be
placed directly in 100% alcohol

39. OTHERS
GLYCOLETHERS
ALCOHOLS
Ethanol
Isopropanol
Butanol
Graded
concentrations of
ethanol are used
for dehydration;
the tissue is
immersed in 70%
ethanol in water,
followed by 95%
and 100%
solutions. Ethanol
ensures total
dehydration,
making it the
reagent of choice
for the processing
of electron
microscopy
specimens
Dioxane
Polyethylene
glycol Acetone
Phenol
When added to
dehydrating
agents, phenol
acts as a
softening agent
for hard tissues
such as tendon,
nail, and dense
fibrous tissue
and keratin
masses. Phenol
(4%) should be
added to each of
the 95% ethanol
stations.

40. CLEARING (DEALCOHOLIZATION)
 Clearing reagents act as an intermediary between the dehydration and
infiltration solutions
 PRINCIPLE: Agent should be miscible with both dehydrating agent and
impregnation agent and acts by replacing dehydrant with a substance
miscible with the embedding medium (Paraffin)

41. The criteria for choosing a
suitable clearing agent are
:
 Rapid penetration of
tissues
 Rapid removal of
dehydrating agent
 Ease of removal by melted
paraffin wax
 Minimal tissue damage
 Low flammability
 Low toxicity

42. Various clearing agents
Xylene: Most commonly used.
Long term immersion results in tissue
distortion therefore tissues should not be left in it for more than
3hours
Toluene
Chloroform: Suitable for nervous tissue
Methyl salicylate: Safe and effective but costly
Benzene : It is carcinogenic.

43.  Volume of clearing agent used is optimally 30-40 times the volume of the
specimen
 The smaller pieces of tissues are cleared in 30minutes to 1hour, whereas
larger tissues (>5mm thick) are cleared in 2-4 hours
 The end point of clearing can be noted by transparent appearance of the
tissue against light

44. INFILTRATION
 It is the process in which the clearing agent is replaced by paraffin or its
substitute that completely fills all tissue cavities
 PRINCIPLE: The wax is infiltrated in the interices of the tissue which
increases the optical differentiation & hardens the tissue & helps in easy
sectioning of the tissue.

45. PARAFFIN WAX:
 Most popular infiltration and embedding medium in histopathology
 It is a polycrystalline mixture of solid hydrocarbons produced during refining
of coal and mineral oils
 Its properties are varied depending on the melting point used, ranging from
47 to 64°C.
 Paraffin wax permeates the tissue in liquid form and solidifies rapidly when
cooled.
 The tissue is impregnated with the medium, forming a matrix and preventing
distortion of the tissue structure during microtomy.

46. MODIFIED PARAFFIN WAXES:
 Paraplast
 Paraplast plus
 Ester waxes
 Polyester wax
 Water soluble wax/PEG: Eliminates dehydration
and clearing, hence lipids and neutral lipids are not
removed and demonstrated in thin section

47.  Tissue processed by
hand requires 6-8
hours in three
changes of wax
whereas with
agitation, 2-4hours in
two baths will suffice
 Successive changes of
tissue in wax redues
clearing agent

48. VACUUM
IMPREGNATION:
It is the impregnation of tissues by
molten medium under reduced
pressure
Rapid, reduces time
Useful for lung tissue and tissue that
contains much air. Also used for
splenic tissue which tend to become
hard in routine processing

49. TISSUE PROCESSORS (Histokinette)
PRINCIPLE: The basic principle for tissue processing requires the exchange of
fluids using a series of solutions for a predetermined length of time in a
controlled environment
Manual or
Automated
TISSUE
PROCESSORS
MOVING
TISSUE TYPE
CAROUSEL
TYPE
LINEAR TYPE
MOVING
REAGENT
TYPE

50. • Characterised by transfer of
tissues, contained within
basket, through a series of
stationary reagents circular
fashion
• Rotary or Carousel is the
most common model
• Provided with 9-10 reagents
and 2-3 wax positions
• Fluids are pumped to and
from retort in which the
tissue casettes remain
stationary
• Depending upon the
model these machines can
process 100-300 casettes
at one time
Tissue transfer processor
Fluid-transfer processor

51. Manual tissue processing :
has stopped in most laboratories.
There are circumstances requiring the tissue sample to be manually
processed, including:
• Power failure or equipment malfunction.
• Large tissue samples requiring more time than can be allocated on an
automated processor.
• Small biopsies, such as transplant specimens needing a rapid diagnosis.

52. Microwave processors
 Now common.
 Shortens processing time from hours to minutes
 Based on principle, that heat peaks up diffusion of liquids in & out of
tissues.
 Microwave exposure stimulates the diffusion of the solutions into the
tissue by increasing the internal heat of the specimen, thus accelerating
the reaction
 Clearing agents are not necessary because the temperature of the final
paraffin step facilitates evaporation of the alcohols from the tissue
 Disadvantages -process is labor intensive because the solutions are
manually manipulated and temperatures must be maintained

53. Alternative rapid processors :
Advances in technology have led to the development of a ‘continuous input
rapid tissue processor’.
The enclosed processor uses microwave technology, vacuum infiltration
and proprietary reagents which are described as being ‘molecular-friendly’.

54. Schedule for processing eyes:
o Must be thoroughly fixed, prior to dissection
o Phenol is added to the lower-percentage alcohols to soften the sclera and
lens
o Chloroform has been used as the clearing agent because it is less harsh
than xylene and causes minimal shrinkage, keeping the retina attached.
o Large tissue cassettes and molds are specifically made for use in processing
eyes.
Rapid processing schedules for small biopsies:
Recently excised endoscopic biopsies and needle biopsies can be adequately
processed in 2–5 hours using heat (37 to 45°C) and vacuum.

55. Tissue restoration:
70% ethanol
70 ml Glycerol
30 ml Dithionite
1 g Tissues
remain in the solution for several hours or overnight.
Processing begins with the dehydrating solutions and continues to
completion.
Tissue may be difficult to section

56. TISSUE EMBEDDING
Embedding involves enclosing of properly processed, correctly oriented
specimens in a support medium that provides external support during
microtomy.
Media used must –
• Fill the matrix within the tissue, supporting cellular components
• Provide elasticity, resisting section distortion while facilitating sectioning

57. Embedding tissue in Paraffin wax:
Most laboratories employ Modular tissue embedding centre
i. Dispensed automatically from a nozzle into a suitably sized mold.
ii. The tissue is oriented in the mold
iii. A cassette is attached, producing a flat block face with parallel
sides.
iv. The mold is then placed on a small cooling area to allow the
paraffin wax to solidify.
v. The quick cooling of the wax ensures a small crystalline
structure, producing fewer artifacts when sectioning the tissue.

58. Modular tissue embedding centre

59. DIFFERENT METHODS OF EMBEDDING:
1. Leuckart or dimmock embedding irons: Two L- shaped pieces of
heavy brass or similar metal, placed in opposing positions and a base is
being formed of 3×2 square inch
2. Paper blocks/boats

60. 3. Peel-A-Away System using disposable
plastic molds
4. Tissue-Tek system

61. ALTERNATIVE EMBEDDING MEDIA:
 Sectioning whole organ such as lung or brain
 Sections are required to be thinner, e.g. lymph nodes
 The use of heat may adversely affect tissues or enzymes
 The infiltrating medium is not sufficiently hard to support the tissue
1. AGAR: Double embedding medium
2. GELATIN: Sections of whole organ in Gough-Wentworth’s organ
sectioning method
3. RESIN: Ultra-thin sectioning for electron microscopy and also for
undecalcified bone

62. 4.CELLOIDIN: Purified form of nitrocellulose.Does not require heat at any
stage of processing so recommended for tissues that can be damaged by
solutions requiring heat
 DOUBLE EMBEDDING AND DOUBLE INFILTRATION
METHODS:
It is the process by which tissues are first embedded or fully infiltratedwith a
supporting medium such as agar, celloidin then infiltrated a second time with
wax in which they are also embedded.The main use of this method is for
cutting sections of delicate tissue and preparing sections from blocks of
tissue of varying consistency eg. eyes where retina is easily detached.

63. Orientation of tissues
 Tubular structures: cross
section of the wall and lumen
should be visible
 Skin biopsies: shave punch or
excisions, cross section of the
epidermis, dermis and
subcutaneous layers must be
visible

64.  Intestine, gallbladder, and other epithelial biopsies: cut
in a plane at right angles to the surface, and oriented so the epithelial
surface is cut last, minimizing compression and distortion of the
epithelial layer.
 Muscle biopsies: sections containing both transverse and
longitudinal planes.
 Multiple pieces of a tissue: are oriented side by side with the
epithelial surface facing in the same direction.

65. FROZEN SECTIONS
At times during performance of surgical procedures, it is necessary to
get a rapid diagnosis of a pathologic process.
The surgeon may want to know if the margins of his resection for a
malignant neoplasm are clear before closing, or an unexpected disease
process may be found
This is accomplished through use of a frozen section.

66.  The piece(s) of tissue to be studied are
snap frozen in a cold liquid or cold
environment (-20 to -70 Celsius).
 Freezing makes the tissue solid enough to
section with a microtome.
 Frozen sections are performed with an
instrument called a cryostat.
 Cryostat is a refrigerated box containing
a microtome with an inside temperature
of -20 to -30 celsius
 The tissue sections are cut and picked up
on a glass slide.
 The sections are then ready for staining.

67. Receiving & labelling
Grossing
Tissue processor Paraffin blocks
Microtomy
Water bath
Stained slides

68. Recognising the common faults in tissue processing
TISSUE FOLDING CHATTER ARTIFACT

69. COVERSLIP ARTIFACT

70. TISSUE MICROARRAY
WHAT IS TISSUE MICROARRAY?
A tissue microarray (TMA) contains many small representative tissue samples
from hundreds of different cases assembled on a single histologic slide, and
therefore allows high throughput analysis of multiple specimens at the same
time.

71.  Battifora (1986) first introduced this concept and then Kononen et al.
(1998) used this mechanism for examining several histological sections at
the same time by arraying them in a single paraffin block
PURPOSE:
 Applications in clinical pathology
 Serves as quality control for new antibodies.
The production of antibodies is an expensive and lengthy process, and TMA is a unique
tool which can aid the streamlining of the cumbersome validation and quality control of
archival tissue as well as daily immunohistochemistry (IHC) controls.

72. Principle of tissue microarray technology
o TMA is essentially a simple mechanical method
o wherein minute cylindrical tissue cores of regular shapes and size are taken
from FFPE “donor” tumor blocks
o and are subsequently arrayed on a “recipient” - TMA block
o To achieve this, a hollow needle is used to remove tissue cores ranging from
o 0.6-2mm in diameter from regions of interest in paraffin embedded tissues
such as clinical biopsies or tumor samples.

73. o These tissue cores are then inserted into a recipient paraffin block in a
precisely spaced, array (grid)pattern.
o The array block can be sectioned approximately 300 times using a
microtome and the sections mounted onto microslides.

74. o The individual slides will have all tissues in the same coordinate
positions.
o The individual slides can now be subjected to a variety of tests,
such as
1. H&E staining to ascertain tissue morphology,
2. Immunostaining for protein expression and
3. analysis of genetic alterations using FISH

75. 1. Prevalence TMAs: To determine the prevalence of a given
alteration in a specific area of interest in a tumor.
2. Progression TMAs contain samples of different stages of one
tumor type and are used to discover associations between tumor
genotype and phenotype. For example, a breast cancer progression
TMA could contain samples of normal breast from patients with
and without a history of breast cancer, different non-neoplastic
breast diseases, ductal and lobular carcinoma in situ, invasive
cancer of all stages, grades and histological subtypes as well as
metastases and recurrences after initially successful treatment.
Types of tissue microarrays

76. 3. Prognosis TMAs : contain samples from tumors available with clinical
follow-up data and represent a fast and reliable method for the evaluation
of clinical importance of new detected disease-related genes.
4. Experimental TMAs: are constructed from cell lines or samples from TMA
archives for testing new antibodies and looking for gene targets.

77. TMAs paraffin blocks produced by
At first, the donor blocks are retrieved and sectioned to produce standard
microscopic slides that are stained with H&E
An experienced pathologist examines the slides to mark the area of interest,
which is commonly an area of cancer after which the samples can be arrayed
A tissue microarray instrument is used to acquire a cylindrical tissue core from
the donor block

78. This core is then placed in an empty paraffin block—the recipient block
 Circular spots that are 0.6
mm in diameter at a
spacing of 0.7-0.8 mm.17
 The surface area of each
sample is 0.282 mm2, or in
pathologists' terms, about
the size of 2-3 high power
fields

79. The core is placed at a specifically assigned coordinate (X-Y guide), which is
accurately recorded, typically on a spreadsheet, such as Microsoft Excel
 Up to 1000 or more tissue samples can be arrayed
 Permit simultaneous analysis of molecular targets at the DNA, mRNA, and
protein levels under identical, standardized conditions on a single glass slide
 Provide maximal preservation and use of limited and irreplaceable archival
tissue samples.

81. Varies, depending on the
purpose of the array, and
needs considerable thought
before tissue transfer occurs
A large number of samples
(high density) can be
arrayed in a 37 × 24 × 5 mm
block and
A smaller number of
samples (low density) in a
24 × 24 × 5 mm block.
GRID DESIGN

82. ADVANTAGE
• Study of tumor biology
• Assessment of new diagnostic tools
• Assessment of prognostic and predictive value
• Quality control and clinical practice
• Amplification of a scarce resource
• Simultaneous analysis of very large numbers of specimens
• Experimental uniformity
• Does not destroy original block for diagnosis and thus conserves
valuable tissue.

83. Tissue heterogeneity and other disadvantages of tissue
microarray
1. Small cores sampled may not be representative of the whole tumor,
particularly in heterogenous cancers such as prostate
adenocarcinoma and Hodgkin lymphoma.
2. With several hundreds of cores in a single block it is vital to keep a
detailed map of each core’s position.
3. Loss of 15% of cores between arraying and the final slide

84. THANK YOU
For a Pathologist, every specimen is a patient, which
we have to make talk and tell about itself…