Histotechnique

2.  Histology is microscopic study of normal tissue of body
 Term histology derived from greek word
histos-means tissue……logy –means study coined
by Mayer in 1819
 Histopathology means sciences of studying
structural changes in human body by
diseases

3.  Histotechnology is concerned with
processing and preparation of tissues in such
a manner that it enables a satisfactory study.
 Histotechnique is that branch of biology
concerned with demonstration of minute
tissue structure in diseases.

4.  Paraffin embedding technique
 Parlodion embedding technique
 Exfoliative cytology
 Fine-needle aspiration cytology
 Fine-needle aspiration biopsy
 Ground section
 Frozen section
 Immunohistochemistry (or IHC)
 Fluorescence technique
 Tissue microarray
 Molecular techniques

5.  The lab should be well illuminated and well-
ventilated.
 Rules and Regulations governing
 formalin and
 hydrocarbonds
 such as xylene
 and toluene.
 Limits set by the Occupational Safety and
Health Administration (OSHA) that should not
be exceeded.
 These limits should be revised and revived to
reduced any mishap.

6.  Check the sharpness of scalpel, scissors and
quality of other ones like ruler, probes weighing
machines.
 Every instrument used in the laboratory should
meet electrical safety specifications and have
written instructions regarding its use.
 Flammable materials may only be stored in
approved rooms and only in storage cabinets that
are designed for this purpose.

7.  Fire safety procedures are to be posted.
Safety equipment including fire
extinguishers,
 fire blankets,
and fire alarms should be within easy
access.
 A shower and eyewash should be readily
available.
 No smoking, eating or movements in the labs
 Use disposable gloves

8.  Laboratory accidents must be documented and
investigated with incident reports and industrial
accident reports.
 Specific hazards that you should know about
include:
 Bouin's solution is made with picric acid. This acid is
only sold in the aqueous state. When it dries out, it
becomes explosive.

9.  Tissue specimens received in the surgical
pathology laboratory have a request form
that lists the patient information and history
along with a description of the site of origin.
 The specimens are accessioned by giving
them a number that will identify each
specimen for each patient.

11.  Tissues removed from the body for diagnosis arrive in
the Pathology Department and are examined by a
pathologist, pathology assistant, or pathology resident.
 Gross examination consists of describing the specimen
and placing all or parts of it into a small plastic
cassette which holds the tissue while it is being
processed to a paraffin block. Initially, the cassettes
are placed into a fixative
 When a malignancy is suspected, then the specimen is
often covered with ink in order to mark the margins of
the specimen. Different colored inks can be used to
identify different areas if needed. When sections are
made and processed, the ink will mark the actual
margin on the slide

25.  Machine processing
 Manual processing

28. 1. FIXATION.
2. DECALCIFICATION (if required).
3. PROCESSING & EMBEDDING.
4. SECTIONING.
5. MOUNTING.
6. STAINING.

29.  Definition –process by which constituents of
cells and tissues are fixed in a chemical so
that they will withstand treatment with
various reagent with minimum loss or
decomposition.
 In simple words—it prevents autolysis of
tissue.

30.  1- To prevent autolysis and bacterial attack.
2- To fix the tissues so they will not change
their volume and shape during processing.
3- To prepare tissue and leave it in a condition
which allow clear staining of sections.
4- To leave tissue as close as their living state
as possible, and no small molecules should be
lost.
 Fixation is coming by reaction between the
fixative and protein which form a gel, so
keeping every thing as their in vivo relation to
each other.

31. Types of fixation
 1-Immersion fixation
 2-Perfusion fixation
 3-Vapour fixation
 4-Spray fixation
 5-Freeze fixation
 6-Microwave fixation
Types of fixatives
 Aldehydes
 Mercurials
 Alcohols
 Picrates
 Oxidizing agents

33.  Inhibition of autolysis
 Hardening of tissue
 Solidification of colloid material
 Optical differentiation
 Effects on staining
 Loss of material during fixation
 Tissue shrinkage

34.  1-Buffer and pH
 2-Temperature
 3-Penetration capacity
 4-Volume change
 5-Agitation
 6-Osmolarity of fixation solution
 7-Concentration of fixation
 8-Duration of fixation

35.  Fixation is best carried out close to
neutral pH, in the range of 6-8.
 Hypoxia of tissues lowers the pH, so there
must be buffering capacity in the fixative
to prevent excessive acidity.
 Acidity favors formation of formalin-heme
pigment that appears as black,
polarizable deposits in tissue.
 Common buffers include phosphate,
bicarbonate, cacodylate, and veronal.
 Commercial formalin is buffered with
phosphate at a pH of 7.

36.  Penetration of tissues depends upon the
diffusability of each individual fixative, which is
a constant.
 Formalin and alcohol penetrate the best, and
glutaraldehyde the worst.
 Mercurials and others are somewhere in
between.
 One way to get around this problem is sectioning
the tissues thinly (2 to 3 mm).
 Penetration into a thin section will occur more
rapidly than for a thick section

37.  The volume of fixative is important.
 There should be a 10:1 ratio of fixative to tissue.
 Obviously, we often get away with less than this,
but may not get ideal fixation.
 One way to partially solve the problem is to
change the fixative at intervals to avoid
exhaustion of the fixative.
 Agitation of the specimen in the fixative will also
enhance fixation.

38. Increasing the temperature, as
with all chemical reactions, will
increase the speed of fixation,
as long as you don't cook the
tissue.
Hot formalin will fix tissues
faster, and this is often the first
step on an automated tissue
processor.

39.  Concentration of fixative should be adjusted down
to the lowest level possible, because you will expend
less money for the fixative.
 Formalin is best at 10%;
 Glutaraldehyde is generally made up at 0.25% to
4%.
 Too high a concentration may adversely affect the
tissues and produce artefact similar to excessive
heat.

40.  Also very important is time interval from of
removal of tissues to fixation.
 The faster you can get the tissue and fix it, the
better.
 Artefact will be introduced by drying, so if
tissue is left out, please keep it moist with
saline.
 The longer you wait, the more cellular
organelles will be lost and the more nuclear
shrinkage and artefactual clumping will occur.

41. Penetrate cells or tissue rapidly
Preserve cellular structure before
cell can react to produce
structural artifacts
Not cause autofluorescence, and
act as an antifade reagent.

42.  Coagulating Fixatives
 Crosslinking Fixatives

43.  Fix specimens by rapidly changing hydration state of
cellular components
 Proteins are either coagulated or extracted
 Preserve antigen recognition often.
 DISADVANTAGE
Advantages
Disadvantages
• Cause significant shrinkage of specimens.
• Difficult to do accurate 3D confocal images.
• Can shrink cells to 50% size (height).
• Commercial preparations of formaldehyde contain
methanol as a stabilizing agent.

44.  Glutaraldehyde
 Formaldehyde
 Ethelene glycol-bis-succinimidyl succinate (EGS)
 Form covalent crosslinks that are determined by
the active groups of each compound

45.  Simple fixative
Eg-Formaldehyde,Glutaraldehyde,Ethyl alcohol
 Compound fixative
Eg-Carnoys fluid,Zenker’s fluid,Bouins fluid
According to action upon cell and tissue
1-Micro-anatomical fixative
To preserve microscopic structure of tissues.
Eg-Formal-saline,Buffered neutral
formalin,Zenker’s fluid

46.  2-Cytological fixative
To preserve intracellular structure.
Eg-Carnoy’s fluid,Clarks fluid,Flemings fluid
 3-Histochemical fixative(freezing-drying
technique)
Eg-Buffered neutral formalin,Cold acetone

47.  According to action
1-Physical methods ------heating
------microwaving
-------freeze drying
2-Chemical methods(biochemical approach)

48.  Tolerant fixative---eg-formalin
 Non tolerant—eg carnoy’s fixative
MOST COMMONLY USED FIXATIVE -----
1-10%formalin
2-10%formal saline
3-10%buffered formalin

49.  The Process of removing calcium salts from the tissue and making them
suitable for sectioning.
 Some tissues contain calcium deposits which are extremely firm and
extremely firm and which will not section properly with paraffin
with paraffin embedding owing to the difference in densities between
densities between calcium and paraffin.
 Bone specimens are the most likely type here, but other tissues may
but other tissues may contain calcified areas as well.
well.
 This calcium must be removed prior to embedding to allow sectioning.
embedding to allow sectioning.
 A variety of agents or techniques have been used to decalcify tissue and
to decalcify tissue and none of them work perfectly.
perfectly.
 Mineral acids,
 organic acids,
 EDTA, and

50. Specimens should be decalcified in
hydrochloric acid/formic acid working
solution 20 times their volume.
Change to fresh solution each day until
decalcification is complete.
 It may take 24 hours up to days or months depending
on size of the specimens.
 Once the decalcification is complete, rinse specimens
in water briefly and transfer to ammonia solution to
neutralize acids left in specimens for 30 minutes.
Wash specimens in running tap water
thoroughly up to 24 hours.
Routine paraffin embedding.

51.  1 –Acid decalcification
 2- Ion exchange resins
 3-Electrical ionization
 4-Chelating methods

52.  1)Aqueous nitric acid(clayden ,1952)
-nitric acid—5-10ml
-distilled water—100ml
 2)Nitric acid –formaldehyde(recommended for urgent
biopsies)
---nitric acid—10ml
----formalin—5-10 ml(added to prevent tissue swelling)
-----distilled water—100ml

53.  3)Gooding and Stewarts fluid(1932)
Formic acid---5ml
Formalin-----5ml
Distilled water----90ml
 4)Trichloroacetic acid
 5)Von Ebners fluid
-Sodium chloride---50ml
-HCl—15ml(added daily 0.5% until
decalcification)
-Distilled water---100ml

55.  Perenyl’s fluid—
10%nitric acid-----40ml
absolute alcohol---30ml
chromic acid(0.5%)—30ml
Excellent cytological preservation are possible-
--
Chemical test cannot be carried out---x-ray
should be used

56.  Nitric and
 Hydrochloric acids
 rapid
 damage cellular morphology,
 so are not recommended for delicate tissues such as bone
marrow.

57. Acetic and Formic acid are better
suited to bone marrow, since they
are not as harsh.
However, they act more slowly on
dense cortical bone.
Formic acid in a 10% concentration
is the best all-around decalcifier.
Some commercial solutions are
available that combine formic acid
with formalin to fix and decalcify
tissues at the same time.

58. EDTA can remove calcium and is not
harsh (it is not an acid)
 but it penetrates tissue poorly and
 works slowly and is
 expensive in large amounts.
Electrolysis has been tried in
experimental situations where calcium
had to be removed with the least tissue
damage.
 It is slow and not suited for routine daily
use.

59.  Most used is EDTA which as ability to bind
calcium forming non-ionized soluble complex
 EDTA works best on cancerous bone
 Agent of choice for electron microscopy
 EDTA solution(hilleman/lee)
----EDTA disodium salt---5.5 g
----Distilled water-----90ml
-----Formalin------10ml

60.  Concentration of active reagent
 Temperature
 Agitation
 Density of bone

62.  X-ray (the most accurate way)
 Chemical testing (accurate)
 Physical testing (less accurate and
potentially damage of specimen)

64.  Insert a pipette into the decalcifying solution
containing the specimen.
 Withdraw approximately 5 ml of the hydrochloric
acid/formic acid decalcification solution from
under the specimen and place it in a test tube.
 Add approximately 10 ml of the ammonium
hydroxide/ammonium oxalate working solution,
mix well and let stand overnight.
 Decalcification is complete when no precipitate
is observed on two consecutive days of testing.
Repeat this test every two or three days.

65. The Physical tests include bending the
specimen or inserting a pin, razor, or
scalpel directly into the tissue.
The disadvantage of inserting a pin,
razor, or scalpel is the introduction of
tears and pinhole artifacts.
Slightly bending the specimen is safer
and less disruptive but will not
conclusively determine if all calcium salts
have been removed.
After checking for rigidity, wash
thoroughly prior to processing.

66. Once the tissue has been fixed, it must
be processed into a form in which it
can be made into thin microscopic
sections.
The usual way this is done is with
paraffin.
Tissues embedded in paraffin, which is
similar in density to tissue, can be
sectioned at anywhere from 3 to 10
microns, usually 6-8 routinely.
The technique of getting fixed tissue
into paraffin is called tissue processing

67. TISSUE PROCESSING
The aim of tissue processing is to embed the tissue in a solid
medium firm enough to support the tissue and give it sufficient
rigidity to enable thin sections to be cut , and yet soft enough not
to damage the knife or tissue.
Stages of processing:
1- Dehydration.
2- Clearing.
3- Embedding.

68.  Wet fixed tissues (in aqueous solutions) cannot
be directly infiltrated with paraffin.
 First, the water from the tissues must be
removed by dehydration.
 This is usually done with a series of alcohols,
say 70% to 95% to 100%.
 Sometimes the first step is a mixture of formalin
and alcohol.
 Other dehydrants can be used, but have major
disadvantages.
 Acetone is very fast, but a fire hazard, so is safe only
for small, hand-processed sets of tissues.
 Dioxane can be used without clearing, but has toxic
fumes

69.  Alcohols –
1)ethanol
2)methanol
3)isopropanol
 Normal and tertiary butanols
glycol-ethers
Ethoxyethanol,polyethylene glycols

70.  Other dehydrants-----1)acetone
2)phenol,beechwood
cresolate and aniline

71.  Ethanol
-clear,colorless flammable
-hydrophillic
Advantages -------non toxic,reliable
Disadvantage------expensive,tissue shrinkage
 Supplied as 99.85%ethanol
 Anhydrous cupric sulphate added to final
ethanol scavanges any water present.

72.  Duration of dehydration should be kept to
the minimum consistent with the tissues
being processed. Tissue blocks 1 mm thick
should receive up to 30 minutes in each
alcohol, blocks 5 mm thick require up to 90
minutes or longer in each change. Tissues
may be held and stored indefinitely in 70%
ethanol without harm

73.  Removal of the dehydrant with a substance
that will be miscible with the embedding
medium (paraffin).
 The commonest clearing agent is xylene.
 Toluene works well, and is more tolerant of
small amounts of water left in the tissues,
but is 3 times more expensive than xylene.
 Chloroform used to be used, but is a health
hazard, and is slow.
 Methyl salicylate is rarely used because it is
expensive, but it smells nice (it is oil of
wintergreen).

74.  Replacing the dehydrating fluid with a fluid that is
totally miscible with both the dehydrating fluid and
the embedding medium.
 Choice of a clearing agent depends upon the
following:
- The type of tissues to be processed, and the type
of processing to be undertaken.
- The processor system to be used.
- Intended processing conditions such as
temperature, vacuum and pressure.
- Safety factors.
- Cost and convenience.
- Speedy removal of dehydrating agent .
- Ease of removal by molten paraffin wax .
- Minimal tissue damage .

75.  Chloroform – tolerant, no effect on RI
 Xylene, Benzene, toluene – rapid, intolerant,
flammable, affects RI
 Esters—n butyl acetate----xylene substitute
 cedar wood oil – tolerant, expensive

76.  Some clearing agents:
- Xylene.
- Toluene.
- Chloroform.
- Benzene.
- Petrol.

77.  When xylene has completely replaced the
alcohol in the tissue, the specimen is ready to be
infiltrated with paraffin.
 It is removed from the xylene and placed in a
dish of embedding paraffin, and the dish is put in
a constant temperature of about 60 °C
 The exact temperature depend upon melting
point of the paraffin used.
 During the course of several hours the specimen
is changed to two or three successive dishes of
paraffin so that all xylene in tissue is replaced by
paraffin

78.  Finally, the tissue is infiltrated with the
embedding agent, almost always paraffin.
 Paraffins can be purchased that differ in
melting point, for various hardnesses,
depending upon the way the histotechnologist
likes them and upon the climate (warm vs.
cold).
 Wax hardness (viscosity) depends upon the
molecular weight of the components and the
ambient temperature.
 High molecular weight mixtures melt at higher
temperatures than waxes comprised of lower
molecular weight fractions.
 Paraffin wax is traditionally marketed by its
melting points which range from 39°C to
68°C.

79.  1802------lce gelatin glycerin
 1879------Nitrocellulose
 1881-------Paraffin
 1950-------Acrylics(L R White)

80.  Paraffin wax embedding
 Water soluble waxes embedding
 Celloidin embedding
 Double embedding
 Gelatin embedding
 Ester wax embedding

82. There are four main mould systems and
associated embedding protocols presently in
use :
1- Traditional methods using paper boats.
2- Leuckart or Dimmock embedding irons or
metal containers.
3- the Peel-a-way system using disposable
plastic moulds and
4- Systems using embedding rings or cassette-
bases which become an integral part of the
block and serve as the block holder in the
microtome.

83. Tissue processing
Embedding moulds:
(A) paper boat;
(B) metal bot mould;
(C) Dimmock embedding
mould;
(D) Peel-a-way disposable
mould;
(E) base mould used with
embedding ring
( F) Cassette

84. General Embedding Procedure
1- Open the tissue cassette, check against worksheet entry to ensure
the correct number of tissue pieces are present.
2- Select the mould, there should be sufficient room for the tissue with
allowance for at least a 2 mm surrounding margin of wax.
3- Fill the mould with paraffin wax.
4 Using warm forceps select the tissue, taking care that it does not cool
in the air; at the same time, correct orientation of tissue in a mould is
the most important step in embedding. Incorrect placement of tissues
may result in diagnostically important tissue elements being missed or
damaged during microtomy.
5- Insert the identifying label or place the labeled embedding ring or
cassette base onto the mould.
6- Cool the block on the cold plate, or carefully submerge it under
water when a thin skin has formed over the wax surface.
7- Remove the block from the mould
.
8- Cross check block, label and worksheet.

87.  Turn on the water bath and check that the
temperature is 35-37ºC.
 Use fresh deionized water (DEPC treated water
must be used if in situ hybridization will be
performed on the sections).
 Blocks to be sectioned are placed face down on
an ice block or heat sink for 10 minutes.
 Place a fresh blade on the microtome.
 Insert the block into the microtome chuck so the
wax block faces the blade and is aligned in the
vertical plane. Set the dial to cut 4-10 µM
sections.
 The blade should be angled 4-6º.

88.  Face the block by cutting it down to the desired
tissue plane and discard the paraffin ribbon.
 If the block is ribboning well then cut another
four sections and pick them up with forceps or a
fine paint brush and float them on the surface
of the 37ºC water bath.
 Float the sections onto the surface of clean
glass slides.
 If the block is not ribboning well then place it
back on the ice block to cool off firm up the
wax.
 If the specimens fragment when placed on the
water bath then it may be too hot.
 Place the slides with paraffin sections in a 65°C
oven for 20 minutes (so the wax just starts to
melt) to bond the tissue to the glass.
 Slides can be stored overnight at room
temperature

90.  A microtome is a mechanical
instrument used to cut biological
specimens into very thin segments for
microscopic examination. Most
microtome use a steel blade and are
used to prepare sections of animal or
plant tissues for histology.

91.  STEEL KNIVES
 NON-CORROSIVE KNIVES FOR CRYOSTATS
 DISPOSABLE BLADES
 GLASS KNIVES
 DIAMOND KNIVES

95.  The embedding process must be reversed
in order to get the paraffin wax out of the
tissue and allow water soluble dyes to
penetrate the sections.
 Therefore, before any staining can be
done, the slides are "deparaffinized" by
running them through xylenes (or
substitutes) to alcohols to water.
 There are no stains that can be done on
tissues containing paraffin.

96.  The staining process makes use of a
variety of dyes that have been chosen for
their ability to stain various cellular
components of tissue.
 The routine stain is that of hematoxylin
and eosion (H and E).
 Other stains are referred to as "special
stains" because they are employed in
specific situations according to the
diagnostic need.

97.  Slides being stained on automated Stainer

98.  Frozen sections are stained by hand, because
this is faster for one or a few individual sections.
The stain is a "progressive" stain in which the
section is left in contact with the stain until the
desired tint is achieved.

100.  The stained section on the slide must be covered
with a thin piece plastic or glass to protect the
tissue from being scratched, to provide better
optical quality for viewing under the microscope,
and to preserve the tissue section for years to
come.
 The stained slide must go through the reverse
process that it went through from paraffin
section to water.
 The stained slide is taken through a series of
alcohol solutions to remove the water, then
through clearing agents to a point at which a
permanent resinous substance beneath the glass
coverslip, or a plastic film, can be placed over
the section.

101.  Exfoliative Cytology – In this method, cells are collected
after they have been either spontaneously shed by the body
("spontaneous exfoliation") or manually scraped/brushed off
of a surface in the body ("mechanical exfoliation"). An
example of spontaneous exfoliation is when cells of
the pleural cavity or peritoneal cavity are shed into the
pleural or peritoneal fluid. This fluid can be collected via
various methods for examination. Examples of mechanical
exfoliation include Pap smears, where cells are scraped from
the cervix with a cervical spatula, or bronchial brushings,
where a bronchoscope is inserted into the trachea and used to
evaluate a visible lesion by brushing cells from its surface
and subjecting them to cytopathologic analysis

102.  Suitable for hard structure like bone and
teeth.
 Similar to paraffin embedding technique and
the only difference is infiltration of
embedding is done in parlodion instade of
paraffin.

103.  It is a diagnostic procedure used to investigate
superficial (just under the skin) lumps or masses.
In this technique, a thin, hollow needle is
inserted into the mass for sampling of cells that,
after being stained, will be examined under
a microscope. There could be cytology exam of
aspirate (cell specimen evaluation, FNAC) or
histological (biopsy - tissue specimen evaluation,
FNAB). Fine-needle aspiration biopsies are very
safe, minor surgical procedures. Often, a major
surgical (excisional or open) biopsy can be
avoided by performing a needle aspiration biopsy
instead. Now a day, this procedure is widely used
in the diagnosis of cancer.

104.  Micrograph of a needle aspiration biopsy
specimen of a salivary gland showing adenoid
cystic carcinoma.

105.  It is the process of detecting antigens (e.g.,
proteins) in cells of a tissue section by exploiting
the principle of antibodies binding specifically
to antigens in biological tissues.
 IHC takes its name from the roots "immuno," in
reference to antibodies used in the procedure,
and "histo," meaning tissue (compare
to immunocytochemistry).
 Immunohistochemical staining is widely used in
the diagnosis of abnormal cells such as those
found in cancerous tumors. Specific molecular
markers are characteristic of particular cellular
events such as proliferation or cell death
(apoptosis).

106.  Immunohistochemistry labels individual proteins,
such as TH (green) in the axons of
sympathetic autonomic neurons

107.  It consist of paraffin blocks in which up to 1000
,separate tissue cores are assembled in array fashion to
allow multiplex histological analysis.
 In the tissue microarray technique, a hollow needle is
used to remove tissue cores as small as 0.6 mm in
diameter from regions of interest in paraffin-embedded
tissues such as clinical biopsies or tumor samples. These
tissue cores are then inserted in a recipient paraffin block
in a precisely spaced, array pattern.
 Sections from this block are cut using a microtome,
mounted on a microscope slide and then analyzed by any
method of standard histological analysis. Each microarray
block can be cut into 100 – 500 sections, which can be
subjected to independent tests. Tests commonly
employed in tissue microarray
include immunohistochemistry, and fluorescent in situ
hybridization. Tissue microarrays are particularly useful in
analysis of cancer samples