Technique on Cytohistology

Intan Chairun Nisa, S.Si., M.Biotech

1. Fixation Technique for cyto and histo
2. Gross-room and Tissue Processing Technique
3. Decalcification for Bony
4. Frozen Section
5. Muscle Biopsy
6. Pulmo Processing
1. Fixation Technique for cyto and histo
2. Gross-room and Tissue Processing Technique
3. Decalcification for Bony
4. Frozen section
5. Muscle Biopsy
6. Pulmo Processing
 Special Fixation for Cytology
Haemorrhagic Fluid  sanguineous inflammatory
exudates. Ex : malignant, tubercular, etc), traumatic
or iatrogenic. The diagnostic efficacy of cytology
can suffer if large numbers of Red Blood Cells
(RBCs) are present in the sample.
• Normal Saline Rehydration technique (NSRT) 
effective lysis RBCs
• Carnoy’s Fixative Acetic acid  well preserved
Ingredients of Carnoy’s fxative
• 95% ethanol (60 ml)
• Chloroform (30 ml)
• Glacial acetic acid (10 ml)

Some of the uses of Carnoy's solution are:

• Enhancing lymph node detection during dissection of cadavers.[6]
• Immunohistochemical fixation and detection of NMDA receptors within
the murine hippocampus
• Applied directly following enucleation for the treatment of keratocystic odontogenic
tumors.[8][9][10]
• Direct application following enucleation (Cuba) for certain kinds of
unicystic ameloblastomas.[11] This appears to decrease the likelihood of recurrence
over enucleation alone.[12] Protein coagulation is thought to limit uptake of these toxic
materials by surrounding tissues, however it is this fact that limits its usefulness as a
treatment agent in general.[13]
• As a fixative for pap smear samples.[14]
• As a fixative agent for both nuclear and mitochondrial DNA in various tissues.[15]
• As a fixative agent to preserve mucus, useful for tissue preparation before staining with
periodic acid-Schiff base
 Histology Physical Fixation Technique
1. Heat Fixation
2. Microwave Fixation = <20 minutes, 55 ⁰C
3. Freeze drying n Freeze subtitution = for solube and
small molecules
 Histology Chemical Fixation Technique
1. Formaldehyde Fixation : Formaldehyde, as 10% neutral
buffered formalin (NBF) is the most common fixative used in
diagnostic pathology.

Advantages:
• The penetration rate of formalin is high.
• Cell morphology well preserved in formalin.
• Cheap, Stable, Easy to make the solution.

Disadvantages;
1. Slow fixation.
2. Formalin reaction is reversible, and it can be removed by washing.
3. Formalin fails to preserve acid mucopolysaccharides.
4. Highly vascular tissue may have dark-brown granules (artefact)
5. Exposure to the skin may cause dermatitis.
6. Chronic inhalation may cause bronchitis.
2. Glutaraldehyde Fixation : aldehyde groups may react with
a wide range of other histochemical targets which include
antibodies, enzymes or proteins  for electron microscopy
because it fixes and preserves the ultrastructure.

Advantages:
1. Better fixation of ultrastructure.
2. Less cell shrinkage.
3. Preservation of protein is better.
4. Good cross-linking with collagen.
5. Less irritating.
Disadvantages;
1. Poor penetration and tissue should be less than 0.5 mm thick.
2. Less stable compound.
3. No lipid fixation.
4. Glutaraldehyde polymerizes above pH 7.5.
5. Costly.
3. Osmium Tetroxide : fixation in electron microscopy. It reacts with
unsaturated bonds in the lipid molecules and fixes them.

Advantages:
1. This is a very good fixative for lipid.
2. It preserves cytoplasmic organelles such as Golgi bodies and mitochondria,
3. Does not make the tissue hard,
Disadvantages:
1. It does not fix the proteins and carbohydrates and therefore it should be used in
combination with other fixative.
2. Osmium tetroxide may react with ribose group and may cause clumping of DNA. This
can be prevented by pretreatment with potassium permanganate or post fxation with
uranyl acetate.
3. Poor penetration in the tissue.
4. Tissue swelling may occur.
5. Toxic and volatizes at room temperature producing harmful vapour. This vapour is toxic
to the eye and respiratory tract.
6. Expensive
4. Bouin’s Fixative
Bouin’s solution contains picric acid. This is an
excellent fixative for glycogen. Bouin's fluid is
especially useful for fixation of gastrointestinal tract.
Bouin’s fixative is not suitable for DNA quantitative
Advantages:
1. It is a good fixative for connective tissue and
glycogen.
2. Rapid penetration rate.
Disadvantages:
1. It produces yellow stain to the tissue
5. Dichromate and chromic acid fixation : used to
prepare neuroendocrine tissues for
staining, especially normal adrenal medulla and
its related tumors, e.g. pheochromocytomas.
6. Fixatives for DNA, RNA and protein analysis :
HEPES-glutamic acid buffer mediated Organic
Solvent Protection Effect (HOPE) fixative
7. Metallic ions as a fixative supplement : Hg2+,
Pb2+, Co2+, Cu2+, Cd2+, [PtCl6]2+ and Zn2+.
Mercury, lead and zinc Fixatives is suggested to be
a better fixative for immunohistochemistry than
formaldehyde alone
 Fixation for individual Tissue
1. Eyes : The globe must be firmly fixed in order
to cut good sections for embedding. may be
fixed in NBF, for approximately 48 hours. Animal
eyes may be fixed in Davidson’s fixative. This
fixative is rapid and gives no artifactual
detachment of the retina. Fixation should not
exceed 24 hours for rodents’ eyes and 48 hours
for larger species.
2. Brain
The problem of fixing a whole brain is to make it
firm enough to investigate the neuroanatomy and
to be able to produce sections for histopathology
and possible immunochemistry. Conventionally
this fixation takes 2-6 weeks. Alcoholic formalin
should not be used for fixation if
immunohistochemistry is to be performed using
biotin-avidin (streptavidin).
3. Breast
Clinical samples should be fixed in 10% NBF for a
minimum of 6–8 hours, to a maximum of 72
hours and should be sliced at 5 mm intervals
after appropriate gross inspection and margin
designation. The time from the tissue acquisition
to fixation should be as short as possible in order
to prevent lysis of clinically important biomarkers
such as estrogen receptors, progesterone
receptors and the human epidermal growth
factor receptor-2 (HER2).
4. Lung
Biopsies are typically fixed in NBF. The lungs from
lobectomy, pneumonectomy and autopsies may
be inflated by, and fixed in NBF instilled under
gentle pressure via the trachea or major bronchi.
Such fixed lungs can be cut within 2-6 hours, and
gross sections are fixed overnight, allowing
sections to be processed and cut the next day.
5. Lymphoid tissue
Special care should be taken with all lymphoid tissue
as many organisms, e.g. Mycobacterium tuberculosis
and viruses may be present in the lymphoreticular
system. There is always a possible infection risk with
such cases. The lymphoid tissue is usually sliced and
a representative sample of fresh tissue taken for
special studies, e.g. microbiology, flow cytometry or
molecular analysis. The rest of the lymph node is
fixed in NBF, although some laboratories fix part of
the tissue in B5 or zinc.
6. Testis
Biopsies of the testes are fixed routinely in NBF.

7. Muscle biopsies
These are received fresh and a portion is
separated for enzyme histochemistry. The tissue
for routine histological assessment is fixed in NBF
and embedded so the fibers of the specimen are
viewed in crosssection and longitudinally. After
processing this is stained with H&E, a trichrome
stain and Congo red if amyloid is suspected.
8. Renal biopsies
Renal core biopsies should be subdivided into three and
each piece should contain an adequate number of
glomeruli, e.g. 6-10 for light microscopy, 1-2 for electron
microscopy and 3-6 for immunofluorescence. Each portion
is then preserved depending upon the method to be used
for
subsequent analysis:
• NBF for routine histology.
• Buffered glutaraldehyde at pH 7.3 for ultrastructural
analysis.
• Snap frozen in isopentane and liquid nitrogen for
immunofluorescence examination
1. Fixation Technique for cyto and histo
2. Gross-room and Tissue Processing Technique
3. Decalcification for Bony
4. Frozen Section
5. Muscle Biopsy
6. Pulmo Processing
 Impregnation
To alter the physical characteristics of paraffin wax, the
following modifications may be done:
1. To increase hardness: addition of stearic acid
2. Reduction of melting point: addition of phenanthrene
3. Improving adhesiveness with tissue and wax: addition of
0.5% of ceresin

The addition of small amount of Dimethyl Sulphoxide

(DMSO) in paraffin wax reduces the infiltration time of the
wax and removes the residual clearing agent. It produces a
homogenous matrix and better support.
1. Fixation Technique for cyto and histo
2. Gross-room and Tissue Processing Technique
3. Decalcification for Bony
4. Frozen Section
5. Lendrum Processing
6. Pulmo Processing
The Methods of Decalcifcation
1. Acid decalcifcation
2. Ion-exchange resin
3. Electrical ionization
4. Chelating solution
5. Surface decalcifcation
Acid Decalcifcation  This is the commonest method of
decalcifcation in routine laboratory process. Acid makes the soluble
calcium salt,
and thereby calcium is removed from the tissue.
The strong acids:
• Hydrochloric acid
• Nitric acid
Weak acids:
• Formic acid
• Trichloroacetic acid

Strong Acid  The strong acids are used in 5–10% concentration.

They are rapid in action.
Aqueous Nitric Acid  This is rapid in action. It does not impair
staining if the end point is not crossed
4.3 Chelating Agents
Chelating agents are
organic substances that
adsorb metals.
Ethylenediaminetetraace
tic acid
(EDTA): EDTA is the most
commonly chelating
agent
4.4 Surface Decalcifcation
In case of surface decalcifcation, the surface layer
of paraffn blocks is inverted in 1% hydrochloric
acid (HCl) for 1 h. The exposed top 30 μm tissue
of the paraffn block is decalcifed. The block
should be washed thoroughly before cutting.
Only the first few paraffin sections are
expected to be free from calcium
To be continue