|
C. Specimen Handling
1. FIX FIRST!
2. Label
D. Routine Turn-
Turn-over of Results
2. Frozen section
= 5-
5-15 minutes
3. Autopsy report
= 1 week
#
¬ March 1997 Board Exams
An autopsy, to be most informative
and helpful, should be done within:
a. 36 hours
b. 72 hours
c. 1 week
d. 24 hours
E. Storage of Specimen, Tissue blocks,
Slides
Slides = Indefinite
$%
&"
¬ Methods:
1. Teasing/Dissociation
selected tissue spx watch glass
(w/ isotonic
salt sol¶n)
Tissues less
than 1 mm in diameter sandwiched
between two
slides or a slide and a cover
slip
¬ Spreading
¬ Pull--apart
Pull
¬ Touch prep/Impression
¬Methods:
4.Frozen Section
=normally used when a rapid diagnosis of
a tissue is required.
= APPLICATIONS:
1. Rapid pathologic diagnosis during
surgery
2. Enzyme histochemistry
3. Demonstration of soluble substances
such as lipids and carbohydrates
4. Immunofluorescent and
immunocytochemical staining
5. Some specialized silver stains,
particularly in neuropathology.
( #%! !' (+ $!,
!*
¬ Cold knife procedure
KNIFE = -40 to -60 C
TISSUE = 5 to -10 C
ENVIRONMENT = 0 to -10 C
" *
¬ If tissues are frozen too hard = chip into
fragments when cut.
Remedy: Surface of the block maybe be
softened by warming slightly with the
finger.
¬ If tissues have not been sufficiently frozen
EXAMINATION
$ -
¬ Preserving fresh tissue for
examination
¬ ~
in
in
histotechnology
¬ m : : to preserve the
morphologic and chemical integrity
of the cell in as life-
life-like manner as
possible.
$ -
¬ p
to
to harden and
protect the tissue from the trauma of
further handling.
$ -
¬ Practical considerations of Fixation:
1. Speed ± the specimen should be
placed in fixative as soon as it is
removed from the body.
2. Penetration ± formalin diffuses into the
tissue at approximately ã
3.Volume
3.Volume ± 20 times the tissue volume
4.Duration of fixation
$ -
¬ Two mechanisms involved in
Fixation:
1. Additive fixation ± whereby the
chemical constituent of the fixative is
taken in and becomes part of the
tissue.
Examples: Formalin
Mercuric fixatives
Osmium tetroxide
$ -
¬ Two mechanisms involved in
Fixation:
2. Non-
Non-additive fixation ± whereby the
fixing agent is NOT taken in, but
changes the tissue composition and
stabilizes the tissue by removing the
bound water attached to hydrogen
bonds of certain groups within the
protein molecule.
2. Temperature
2
±
± Õ
ÀFormalin heated at 60 C
= sometimes used for rapid fixation
of very urgent biopsy specimens
4. Osmolality
5. Concentration
6. Duration of Fixation
$ -
I. Aldehyde fixatives
1. Formaldehyde
2. 10% Formol-
Formol-Saline = p
3. 10% BNF (Buffered Neutral
Formalin) =
!"p#""2 $pp"
~$% $&"
$ -
4. Formol-
Formol-Corrosive (Formol-
(Formol-Sublimate)
5. Glutaraldehyde = preserves plasma
protein better.
6. Formol-
Formol-calcium = for the preservation
of lipids
$ -
II. Metallic fixatives
1. Mercuric Chloride
= may produce black granular
deposits on tissues
a. Zenker¶s Fluid (with glacial
acetic acid)
b. Zenker-
Zenker-Formol (Helly¶s Sol¶n)
$ -
II. Metallic fixatives
1. Mercuric Chloride
c. Heidenhain¶s SuSa ±
'(
d. Schaudinn¶s fluid
e. Ohlmacher¶s fluid
f. Carnoy-
Carnoy-Lebrun fluid
g. B-
B-5 fixative
$ -
II. Metallic fixatives
2. Chromate
a. chromic acid
b. potassium dichromate
c. Regaud¶s (Moller¶s)
d. Orth¶s fluid ± for Rickettsia and
other bacteria
- for study of early
degenerative process
$ -
II. Metallic fixatives
3. Lead fixatives ±are generally for
ACID MUCOPOLYSACCHARIDES
(for example: Umbilical Cord/
Wharton¶s jelly)
$ -
III. Picrate fixatives
' '
)'
)
)
-picrates are formed upon protein;
precipitates are soluble in water;
hence tissues must be first rendered
insoluble by direct immersion in 70%
ETOH
$ -
III. Picrate fixatives
-picrate fixatives MUST NEVER be
washed in water before dehydration.
V. Alcoholic Fixatives
¬ produces
(
,
-
¬ Prevention: add saturated aqueous
mercuric chloride
¬ Remedy: Black osmic oxide crystals
may be dissolved in
)
)..
¬ Precaution: may cause conjunctivitis
or blindness.
"" !& $&.
¬ FLEMMING¶S SOL¶N
$ -
VII. Tricholoroacetic acid
Àoverheating (above 65 C)
-vacuolation
-overstained cytoplasm
-pyknotic nuclei
$ -
FIXATIVES FOR E.M.
1. Glutaraldehyde
2. Platinic chloride (PtCl3)
3. Platinic Chloride-
Chloride-formalin
(Zamboni¶s fixative)
4. Gold chloride (AuCl)
5. Osmium tetroxide
6. 10% BNF = acceptable but not
recommended
`
¬ A procedure whereby calcium or lime
salts are removed from tissue
FOLLOWING FIXATION
2. 5-
5-10% aqueous sodium
bicarbonate solution for several
hours.
¬ Adequate water rinsing can usually
be accomplished in 30 minutes for
small samples and 1-
1-4 hrs. for larger
specimens.
!'
¬ For unduly hard tissues that may
damage the microtome knives.
¬ 4% aq. phenol.
¬ Molliflex
¬ 2% HCl
in increasing strengths.
¬ Increasing strengths = all the
!=
!=
ethanol + small amt. of methanol
used in the same way as ETOH
|0
= many of the
=
processing methods for use in a
microwave oven recommend this
agent.
6. Triethyl phosphate
. ! % + +*
1.) 4% phenol + each 95% ETOH
baths
ÀWater(present) =
anhydrous copper sulfate =
turns to blue
(100% ETOH should be changed.)
""12
¬ WHATEVER dehydrating agent is
used, the amount in each stage
should not be less than 10 times the
volume of the tissue in order to
ensure complete penetration of the
tissue by the dehydrating solution.
¬ Also known as DEALCOHOLIZATION
2. Toluene
3. Chloroform
NO DEALCOHOLIZATION
3.) Gelatin
4.) Plastic
#
¬ Paraffin ± simplest, most common
and the BEST infiltrating/embedding
medium.
- is NOT recommended for
fatty tissues ( the dehydrants and
clearing agents used in the process
dissolve and remove fat from the
tissues).
''3
¬ After clearing, tissue is submerged in
2 or more changes of melted paraffin
wax.
epoxy
polyester
acrylic
#
¬ CASTING OR BLOCKING
½ Examples:
½Ehrlich¶s ±slowly ripened
½Delafield¶s ±slowly ripened
½Mayer¶s ± sod. Iodate
½Gill¶s ± sod. Iodate
½Harris ± mercuric oxide
II. Iron Hematoxylins
> iron salts are used as oxidizing agents
and mordant
> examples:
1. Weigert¶s ± Ferric chloride
-for mucles/connective tissue
fibers
2. Heidenhain¶s-
Heidenhain¶s- Ferric ammonium
sulfate
-for mitochondria, muscle
striations, chromatin, and myelin
III. Tungsten Hematoxylin
> Mallory¶s PTAH (Phophotungtic
Acid Hematoxylin)
-for staining muscle
striations
H and E staining Steps:
XYLOL ( 2 CHANGES)
WATER
À
À
-place in Weigert¶s iodine
-wash in dist. Water
-remove iodine with 5% sod.
Thiosulfate
-wash in running water
-proceed with stain
Stain with Harris/ Ehrlich¶s/Delafield¶s
Xylol/xylene
Acid Alcohol
" +
Blueing step
Xylol
Eosin
Bismarck brown
Lithium carbonate
Phosphotungstic acid
Light green SF
(36,50,65)