HISTOPATHOLOGY AND CYTOLOGY TECHNIQUES

 Histopath Reports (Triplicate copies)  Microscopes used: Phase contrast or

o Surgical Pathology Bright field
o Cytopathology Report
B. Squash Preparation (Crushing)
o Autopsy Report
 Signatories  Small pieces of tissues (<1mm) are
o Request forms – PATIENT’S placed in a slide and forcibly
DOCTOR compressed with another slide or cover
o Result forms – PATHOLOGIST glass
 Specimen Handling
C. Smear Preparation
o We FIX the specimen FIRST before we
label.  Cellular materials are spread lightly over
 Routine Turn-over of Results a slide by means of a wire loop,
o Surgical Pathology = 24 hours applicator or another slide
o Cytopathology Report = 5-15 minutes  Especially useful in CYTOLOGICAL
o Autopsy Report = 1 week EXAMINATIONS, particularly for
 Storage of specimen, tissue block and CANCER DIAGNOSIS
slides
o Specimen: 1 month to 1 year SMEARING METHODS
o -Tissue blocks: 3 years to 10 years a. Streaking
o -Slides: Indefinite b. Spreading – recommended for fresh
sputum and bronchial aspirates, also for
Tissue Examination thick mucoid secretions
c. Pull-apart – for serous fluids,
1. Fresh Tissue Examination concentrated sputum, enzymatic lavage
2. Preserved Tissue Examination from GI tract, and blood smears
d. Touch preparation (Impression smear)

FRESH TISSUE EXAMINATION

D. Frozen Section
Methods of Fresh Tissue Examination
 Used when a RAPID DIAGNOSIS is
A. Teasing or Dissociation required
B. Squash Preparation (Crushing)  Recommended for LIPIDS and
C. Smear Preparation NERVOUS TISSUE ELEMENTS
D. Frozen Section
Applications:
 Rapid pathologic diagnosis during
A. Teasing or Dissociation surgery
 Enzyme histochemistry
 specimen is immersed in watch glass  Demonstration of soluble substances
with isotonic salt solution, separated and such as lipids and carbohydrates
examined under the microscope (either  Immunofluorescent and
stained or unstained) immunocytochemical staining
  Some specialized silver stains, o Light microscopy – 2 cm2
particularly in neuropathology o Thin
o Light microscopy – no more than
0.4 cm
FIXATION
Two mechanisms involved in Fixation: Main factors involved in fixation:

1. Additive fixation  Osmolality

o whereby the chemical constituent of the o Hypertonic solutions = cell
fixative is taken in and becomes part of shrinkage
the tissue by forming CROSSLINKS or o Isotonic as well as hypotonic = cell
MOLECULAR COMPLEXES and swelling & poor fixation •
GIVING STABILITY TO THE SLIGHTLY HYPERTONIC (400
PROTEIN to 450 mOsm) = BEST RESULTS
o Examples: Formalin, Mercuric fixatives,  Concentration
Osmium tetroxide o Formaldehyde is normally used as
2. Non-additive fixation 10% solution
o whereby the fixing agent is NOT taken o Glutaraldehyde = 3%
in, but alters the tissue composition and  Duration of Fixation
stabilizes the tissue by removing the o Primary fixation in buffered
bound water attached to H-bonds of formalin = 2 to 6 hours
certain groups within the protein o Prolonged fixation may cause
molecule. shrinkage and hardening of tissue
o Example: Alcoholic fixatives o Electron microscopy = 3 hours

Main factors involved in fixation: PRACTICAL CONSIDERATIONS OF

FIXATION:
 pH
o satisfactory fixation occurs  Speed – specimen should be placed in
between pH 6 and 8 fixative as soon as it is removed from the
 Temperature body (to prevent autolysis and
o Surgical specimens – ROOM putrefaction)
TEMPERATURE  Penetration – formalin diffuses into tissue
o Tissue processors – 40°C at a rate of ~1mm/hr
o Electron microscopy and  Volume – traditionally, amount of fixative
histochemistry – 0 to 4°C has been 10 to 25x the volume of tissue;
o Heat for bacteriology and blood recently, fixative volume should be 20x
films the tissue volume
o Formalin at 60°C for very urgent  Duration of fixation
biopsy specimens REMEMBER:
o Formalin at 100°C to fix tissues
with tuberculosis  Fixative volume should be 20x the tissue
 Thickness of section volume!
o Small
o Electron microscopy – 1 to 2 mm2
Factors that retard fixation of tissues are the  LIPID (for cryostat)–mercuric chloride
following: and potassium dichromate
 PHOSPHOLIPIDS – aldehydes, Baker’s
 Size and thickness of the tissue
formol-calcium
specimen – larger tissues require more
 CHOLESTEROL – digitonin
fixatives and longer fixation time
 CARBOHYDRATES – alcoholic
 Presence of mucus – prevents complete
fixatives
penetration of fixative
 PROTEIN – neutral buffered formol
 Presence of fat – fatty tissues should be
saline or formaldehyde
cut in thin sections and fixed longer
 GLYCOGEN–alcohol-base such as
 Presence of blood – tissues containing
Rossman’s fluid or cold absolute alcohol
large amount of blood should be flushed
 For ELECTRON CYTOCHEMISTRY,
out with saline by arterial cannulization
two aldehyde fixatives are useful
before fixing
o KARNOVSKY’S
 Cold temperature – inactivates enzymes
PARAFORMALDEHYDE
 Agitation (when automatic or
GLUTARALDEHYDE
mechanical tissue processing is used)
o ACROLEIN
accelerates fixation.

Types of Fixatives According to Composition ALDEHYDE FIXATIVES

and Action
 Formaldehyde (Formalin)
1. According to composition  10% Formol-Saline
A. Simple Fixatives  10% Neutral Buffered Formalin or
A.1. Aldehydes Phosphate Buffered Formalin (pH = 7)
A.2. Metallic fixatives  Formol-Corrosive (Formol-Sublimate)
A.3. Picrate fixatives TYPES OF FIXATIVES ACCORDING TO
A.4. Heat ACTION
B. Compound Fixatives MICROAN CYTOL CYTO HISTOC
a. According to action ATOMIC OGICA LOGIC HEMICA
I. Microanatomical Fixatives L AL L
II. Cytological Fixative (NUCLE (CYTO
b. Nuclear Fixatives AR) PLAMI
I. Cytoplasmic Fixatives C)
II. Histochemical Fixatives -10% formol - - -10%
saline Flemming’ Flemming’ formol
 REMEMBER: -10% NBF s s fluid saline
o Nuclear fixatives contain - -Carnoy’s without -Absolute
Heidenhain’s -Bouin’s acetic acid ethyl
GLACIAL ACETIC ACID (has
Susa - -Helly’s alcohol
affinity for nuclear chromatin) -Formol Newcomer Formalin -Acetone
o Cytoplasmic fixatives must sublimate ’s with post -
NEVER contain glacial acetic (formol - chroming Newcomer’
acid (destroys mitochondria and corrosive) Heidenhai -Regaud’s s
Golgi bodies) -Zenker’s n’s Susa -Orth’s
solution fluid
-Zenker-
FIXATIVES FOR SOME SUBSTANCES Formol
(Helly’s)
-Bouin’s
solution
-Brasil’s
solution
 Alcoholic Formalin (Gendre’s) Fixative o Used for routine light microscopic
 Glutaraldehyde work
o Buffered glutaraldehyde followed by
MUST REMEMBER:
secondary fixation in osmium
A. 10% FORMALIN tetroxide is satisfactory for electron
o Made from formaldehyde (gas microscopy
produced by oxidation of METHYL
ALCOHOL)
o Commercially available in 35 to 40% METALLIC FIXATIVES
gas by weight
o Pure stock solution of 40% formalin is  Mercuric Chloride
UNSATISFACTORY for routine  Chromate
fixation  Lead fixatives
o Commonly used as a 4% solution
(giving 10% formalin for tissue A. Mercuric Chloride
fixation)  Zenker’s Fluid (with glacial acetic acid
o Prolonged storage of formaldehyde,  Zenker-Formol (Helly’s solution)
 Heidenhain’s Susa
especially at very low temperature,
 B-5 fixative
may induce precipitation of white
 Carnoy-Lebrun fluid
paraformaldehyde deposits and
 Schaudinn’s fluid
produce turbidity although this, in
 Ohlmacher’s fluid
itself, does not impair the fixing
property of the solution.
o Precipitates may be removed by MUST REMEMBER FOR Mercuric
filtration or by addition of 10% Chloride Fixatives
methanol.
o Concentrated solutions MUST  Frequently used in saturated aqueous
NEVER be neutralized since this solutions of 5 to 7%
might precipitate VIOLENT  Widely used as secondary fixative
EXPLOSIONS  Contain BLACK PRECIPITATES of
MERCURY (except Susa)
B. 10% Formol-Saline  Mercury deposits are removed by
o CNS and general post-mortem tissues treating the specimen with 0.5%
for histochemical examination IODINE SOLUTION in 70%
o 10% Neutral Buffered Formalin or ETHANOL (alcoholic iodine solution)
for 5 to 10 minutes
Phosphate Buffered Formalin (pH =
 Sections are rinsed in water, decolorized
7)
for 5 minutes in 5% sodium thiosulfate
o Surgical, post-mortem and research
and washed in running water
specimens
 ROUTINE FIXATIVE for TISSUE
PHOTOGRAPHY
C. Formol-Corrosive (Formol-Sublimate)
o Routine post-mortem tissues USES
o Alcoholic Formalin (Gendre’s)
 Zenker’s Fluid (with glacial acetic acid)
Fixative
– liver, spleen, CT fibers, nuclei
D. Glutaraldehyde
  Zenker-Formol (Helly’s solution) – ALCOHOLIC FIXATIVES
pituitary gland, bone marrow, spleen
and liver  Used in 70 to 100% concentration (less
 Heidenhain’s Susa – tumor biopsies; concentrated solutions will produce lysis
excellent cytological fixative of cells)
 B-5 fixative – for bone marrow biopsies  Causes POLARIZATION (glycogen
granules move towards the poles or ends
of cells)
CHROMATE FIXATIVES
 CHROMIC ACID A. 100% Methanol – dry and wet smears, blood
 POTASSIUM DICHROMATE- smears and BM tissues
preserves lipid and mitochondria B. 95% Isopropyl alcohol – touch preparations
 REGAUD’S (MOLLER’S) C. Ethyl alcohol- slow penetration
 ORTH’S FLUID - for study of early D. Carnoy’s fluid – MOST RAPID
degenerative processes and tissue FIXATIVE (1 to 3 hours), may be used to
necrosis; demonstrates RICKETTSIAE fix brain tissue for diagnosis of rabies,
and other bacteria preserves Nissl granules.
 Lead fixatives – are generally for ACID E. Newcomer’s fluid – acts as both nuclear and
MUCOPOLYSACCHARIDES (for histochemical fixative.
example: Umbilical Cord/Wharton’s F. Gendre’s- recommended for glycogen
jelly) fixation.

OSMIUM TETROXIDE FIXATIVES

PICRATE FIXATIVES
 Should be kept in a dark-colored,
 Highly explosive when dry
chemically clean bottle to prevent
 Will produce excessive yellow staining
evaporation and reduction by sunlight or
of tissues
organic matter
 Picrates are formed upon protein;
 Requires only 5 to 10x that of tissue
precipitates are soluble in water; hence
volume
tissues must be first rendered insoluble
 INHIBITS HEMATOXYLIN and
by direct immersion in 70% ETOH
makes counterstaining difficult.
 Picrate fixatives MUST NEVER be
 PRODUCES BLACK PRECIPITATE
washed in water before dehydration.
(OSMIC OXIDE)
 If deposited in cornea, blindness will
occur
 Bouin’s Solution – for fixation of embryos
and pituitary
 Brasil’s Alcoholic Picroformol – less messy
Formation of black deposits may be prevented
than Bouin’s
by adding SATURATED AQUEOUS
MERCURIC CHLORIDE

GLACIAL ACETIC ACID Black osmic oxide crystals may be dissolved in

cold water
 fixes nucleoprotein
A. Flemming’s Solution – most common  50-70% alcohol is used to wash out
chrome-osmium acetic acid fixative used excess amount of picric acid (Bouin’s
B. Flemming’s without acetic acid solution).

FIXATION IS RETARDED BY
TRICHLOROACETIC ACID
 Larger tissues
– may also be used as a weak
 Presence of mucus
decalcifying agent
 Presence of fat
ACETONE  Presence of blood
 Cold temperature
– used for the diagnosis of Rabies
HEAT FIXATION
FIXATION IS ENHANCED BY
– Involves thermal coagulation of tissue
proteins for rapid diagnosis  Smaller and thinner tissues
 Agitation

FIXATIVES FOR ELECTRON

MICROSCOPY
1. Glutaraldehyde
2. Platinic chloride (PtCl3)
3. Platinic Chloride-formalin (Zamboni’s
fixative)
4. Gold chloride (AuCl)
5. Osmium tetroxide
6. 10% BNF

Washing-out
is the process of removing excess
fixative from the tissue after fixation in
order to improve staining and remove
artifacts from the tissues.

 Alcoholic iodine is used to remove

excessive mercuric fixatives.
 Tap water is used to remove excess
chromates from tissues fixed in Helly’s,
Zenker’s, and Flemming’s solutions,
excess formalin and excess osmic acid.