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By: Rene Jesus Alfredo R.


Dinglasan, RMT
   
!"!
A. Histopath Reports
1. Surgical pathology
2. Cytopathology report
3. Autopsy report
‡ Ô 
      
Three copies =
Doctor
Patient
File
#

  
¬March 1992 Board Exams:
Forensic and anatomic pathology
report should be kept in the
laboratory for a period of:
a. one year
b. two years
c. six years
d. permanently
#

  
¬ March 1998 Board Exams
In most private hospitals, the
histopathologic report is typed in:
a. Four copies
b. Triplicate
c. Duplicate
d. One copy
B. Signatories

1. Request Forms = patient¶s doctor

2. Result Forms = Pathologist

C. Specimen Handling

1. FIX FIRST!

2. Label
D. Routine Turn-
Turn-over of Results

1. Surgical pathology and cytology


= 24 hours

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

Specimen = 1 month to 1 year

Tissue Blocks = 3 years to 10 years

Slides = Indefinite
 $% 
&"
¬ Methods:
1. Teasing/Dissociation
selected tissue spx watch glass
(w/ isotonic
salt sol¶n)

Microscopes used: Phase


contrast/Bright
field
¬ Methods:
2. Crushing/ Squash preparation

Tissues less
than 1 mm in diameter sandwiched
between two
slides or a slide and a cover
slip

a vital stain may be used


¬ Methods:
3. Smear prep = process of examining
sections or sediments
= cellular materials are
spread lightly over a slide by means
of a wire loop/ applicator stick/
another slide.
'' " (( %)
 *
¬ Streaking

¬ 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

= thick and crumble


= block may come away from
the stage.
Remedy: More bursts of carbon dioxide gas
should then be given to refreeze the block.
( #%!  !' (+ $!,
!*
¬ Cryostat procedure (Cold Microtome)
optimum working temp. = -18 to -20 C
CRYOSTAT ± a refrigerated cabinet in
which a modified microtome is housed.
All the controls to the microtome are
operated from outside the cabinet.
Presently, the r rr   is the
type of choice.
!""!  #%!  !'
$,+
¬ Liquid Nitrogen
¬ Isopentane cooled by liquid nitrogen

¬ Carbon dioxide gas

¬ Aerosol sprays = adequate for

freezing small pieces of tissue


EXCEPT muscle.
PRESERVED TISSUE

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.

Example: Alcoholic fixatives


$ - 
¬ Main factors involved in fixation:
1. Hydrogen ion concentration (pH)
satisfactory fixation = 


2. Temperature

  

   2  

     

±

 ± Õ
ÀFormalin heated at 60 C
= sometimes used for rapid fixation
of very urgent biopsy specimens

ÀFormalin heated at 100 C


= can be used to fix tissues with TB
$ - 
3. Thickness of section

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.

TISSUES 70% ETOH 5% Sod.


Thiosulfate

wash in running water


$ - 
III. Picrate fixatives
A. Bouin¶s Solution ±     
 

B. Brasil¶s Alcoholic Picroformol ± less


messy than Bouin¶s
$ - 
IV. Glacial Acetic Acid
= fixes nucleoprotein

V. Alcoholic Fixatives

 
  

m 2$* $


¬ Polarization ± glycogen granules move towards
the poles or ends of cells.

1. Methanol ± blood smears and BM


tissues
2. Ethanol

3. Carnoy¶s fluid ± þp2 m$+


~$% $&"
( Fixation time: 1 to 3 hours )
4. Alcoholic Formalin ( Gendre¶s Fixative)
-useful in preserving  
5. Newcomer¶s fluid
$ - 
VI. Osmium Tetroxide Fixatives

- should be kept in a dark


dark--colored,
chemically clean bottle to prevent
evaporation and reduction by
sunlight or organic matter.

- inhibits hematoxylin and makes


counterstaining difficult.
  "" !&  $&.

¬ 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

¬ FLEMMING¶S W/O ACETIC ACID

 
  

   
 

$ - 
VII. Tricholoroacetic acid

VIII. Acetone ± used for the Dx of


Rabies

IX. Heat Fixation


± direct flaming fixation

- microwave fixation (optimum temp. 45-


45-55 C)

Àunderheating ± poor sectioning

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

¬ Should be done after fixation and


before impregnation
` 
¬ More concentrated acid solutions
decalcify bone more rapidly but are
more harmful to the tissue.
¬ High concentrations and greater
amount of fluid will increase the
speed of the process.
¬ The recommended ratio of fluid to
tissue volume for decalcification is 20
to 1.
¬ Heat will serve to hasten decalcification
BUT it also increases the damaging
effects on tissues.
¬ At 37 C = impaired nuclear staining of
Van Gieson¶s stain for collagen fibers.
¬ At 55 C = tissue will undergo complete
digestion within 24-
24-48 hours.
¬ Optimum temperature = RM TEMP (18- (18-
30 C)
¬ The ideal time required for
decalcifying tissue is 24-
24-48 hours.
¬ Dense bone tissues usually require
up to 14 days or longer in order to
complete the process.
'+ +
¬ Nitric acid ± MOST COMMON
examples: m
 


 acts as
BOTH tissue softener and
decalcifying agent.
m 


Ô



MOST RAPID
DECALCIFYING AGENT!
'+ +
¬ Formic acid ± BOTH fixative and
decalcifying agent
¬ 5% formic acid is considered to be
the BEST GENERAL DECALCIFYING
AGENT
¬ Formic acid is recommended for
small pieces of bones and teeth.
'+ +
¬ Hydrochloric acid
> Von Ebner¶s fluid ± recommended
for teeth and small pieces of bones
and teeth.
!//'!
!
¬ After decalcification is complete, acid
can be removed from tissues or
neutralized chemically by immersing
the decalcified bone in either:
1. saturated lithium carbonate sol¶n.

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

¬ 1% HCl in 70% alcohol

Note: Tissues immersed in Molliflex


may appear swollen and soapy.
(Does not affect normal processing)

|0 
¬ Aim: to remove fixative and water
from the tissue and replacing them
with dehydrating fluid in preparation
for impregnation.
¬ Dehydrating fluids are generally used

in increasing strengths.
¬ Increasing strengths = all the

aqueous tissue fluids are removed


but with little disruption to the tissue
due to diffusion currents.

|0 
¬ For delicate tissues, particularly
 
    , , it
is recommended to start processing
with 30% ethyl alcohol
alcohol..

|0 
COMMONLY USED DEHYDRATING AGENTS:
1.) Alcohol ± MOST COMMON
  = for routine dehydration of
tissues.
= BEST DEHYDRATING
AGENT.

 = employed for blood


 =
and tissue films

|0 
  =utilized
 =utilized in plant and
animal microtechniques.

 
  


     !=
  !=
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.

2. Acetone ± BOTH fixative and


dehydrating agent.

3. Dioxane (Diethylene dioxide) ±


BOTH dehydrating and clearing agent

|0 
4. Cellosolve (Ethylene glycol
monoethyl ether) ± BOTH
dehydrating and clearing agent

5. THF (Tetrahydrofuran) ± BOTH


dehydrating agent and clearing agent

6. Triethyl phosphate
 . ! % + +*
1.) 4% phenol + each 95% ETOH
baths

acts as a tissue softener for


hard tissues such as tendons, nails,
or dense fibrous tissues.
 . ! % + +
2.) Anhydrous copper sulfate
= can act as BOTH dehydrating
agent and an indicator of water content
of the last bath (100% ETOH).

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

¬ Process of replacing the dehydrating


fluid with a fluid that is miscible with
BOTH the dehydrating fluid and the
impregnating/embedding medium.
medium.

 
Clearing agents suitable for routine
use:
1. xylene/xylol

2. Toluene

3. Chloroform

4. Methyl benzoate and methyl


salicylate
5. Cedarwood oil and clove oil

 
6. Citrus fruits oils
7. Trichlorethane and petrol
8. Benzene
9. Aniline oil
10. Carbon tetrachloride

 
¬ Exemption to the rule:
GLYCERIN AND GUM SYRUP

NO DEALCOHOLIZATION

These clearing agents merely make


the tissue clearer!
#
  
¬ Also known as INFILTRATION

¬ Process of replacing the clearing


agent with the infiltrating medium.

¬ The medium used to infiltrate the


tissue is usually the same medium
used for embedding.
#
  
Four types of tissue impregnation and
embedding media:
1.) Paraffin wax

2.) Celloidin (Collodion)

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.

¬Temperature of paraffin oven =


55--60 C
55
m  
 

   
"#$ m
   "
  
%   !
''3
¬ Wax with melting point = 56 C is
normally used for routine work.

¬ If the lab temperature = 20-


20-24 C

paraffin wax MP to use: (54-


(54-58 C)
''3
¬ If the lab temp. = 15-
15-18 C

paraffin wax MP to use: 50-


50-54 C
''3
¬ When wax has been reused, some
water is mixed with it.
¬ If excessive water accumulates, this
may impair the impregnating
capacity of the medium.
¬ To remove excess water = heat the
wax to 100-
100-105 C
¬ Paraffin wax may be used twice only!
SUBSTITUTES FOR PARAFFIN WAX
1. Paraplast = MP: 56-
56-57 C
= mixture of highly
purified paraffin and synthetic plastic
polymers
= more elastic and
resilient than paraffin

= for large dense tissue


blocks such as bones and brain
SUBSTITUTES FOR PARAFFIN WAX
2. Embeddol = MP: 56-
56-58 C
=less brittle and less
compressible than paraplast.

3. Bioloid = recommended for


embedding eyes.

4. Tissue Mat = a product of paraffin,


containing rubber, with the same
property as paraplast.
SUBSTITUTES FOR PARAFFIN WAX
5. Ester Wax = MP: 46-46-48 C
= harder than paraffin
=not soluble in water
=soluble in 95% ETOH
and other clearing agents.
=can be used for
impregnation without prior clearing of the
tissue.
=sectioning of ester wax-
wax-
impregnated tissues should be done on a
sliding or sledge type microtome due to
the relative hardness of the wax.
SUBSTITUTES FOR PARAFFIN WAX
6. Water-
Water-soluble waxes = MP: 38-
38-42 C
or 45-
45-56 C
= mostly polyethylene glycols

ÀMost commonly used: CARBOWAX


¬ Carbowax ± soluble and miscible with
water (hence does not require
dehydration and clearing of the
tissue).
- tissues are fixed,
washed out and transferred directly
into melted carbowax.

- suitable for many


enzyme histochemical studies.
#
  
¬ Celloidin (Collodion) ± purified form
of nitrocellulose
=suitable for
specimens with large hollow
cavities, hard and dense tissues
(bones and teeth), large tissue
sections of the whole embryo.
! 3
Two methods for celloidin
impregnation:
1. Wet Celloidin ± recommended for
bones, teeth, large brain sections
and whole organs.
2. Dry Celloidin ± preferred for
processing of whole eye sections.
! 3
¬ L.V.N. (Low Viscosity Nitrocellulose)
is another form of celloidin
¬ It is soluble in equal concentration of
ether and alcohol, with a lower
viscosity, allowing it to be used in
higher concentrations and still
penetrate tissues rapidly.
#
  
¬ Gelatin ± rarely used except when
dehydration is to be avoided.
- used when tissues are for
histochem and enzyme studies.
- embedding medium for
delicate specimens and frozen
sections because it prevents
fragmentation of tough and friable
tissues when frozen sections are cut.
3
¬ It is water-
water-soluble

¬ Does not require dehydration and


clearing
#
  
¬ Plastic/Resin ±classified into:

epoxy

polyester

acrylic

#
 
¬ CASTING OR BLOCKING

¬ Process by which the impregnated tissue


is placed into a precisely arranged position
in a mold containing a medium which is
then allowed to solidify.
¬ V Ô&&VÔ
Ô&&VÔprocess by which a tissue
is arranged in precise positions in the
mold during embedding, on the microtome
before cutting, and on the slide before
staining.

#
 
¬ Temperature of melted paraffin used
for embedding = 5-10 C  its
MP.
¬ To solidify embedded tissue = cooled
rapidly in a ref (-
(-5 C) or immersed in
cold water.
¬ The surface of the section to be cut

should be placed parallel to the


bottom of the mold in which it is
oriented.
 ## 
¬ Process of removing excess wax
after embedding.
¬ Excess wax is cut off from the block
to expose the tissue surface in
preparation for actual cutting.
¬ Knife/blade may be used

  
¬ CUTTING OR MICROTOMY
¬ The process by which a processed
tissue is cut into uniformly thin slices
(sections) to facilitate studies under
the microscope.
¬ 4-6 u in thickness for routine
histologic procedure.
¬ 10
10--15 u for frozen section.
section.
¬ 0.5 u for electron microscopy.

  
KINDS OF MICROTOMES:
1. Rocking Microtome (Cambridge
Rocking Microtome)
Àinventor: Paldwell Trefall in 1881
Àsimplest among the microtomes
Àdisadvantage: difficulty in
reorienting the block.

  
2. Rotary/Minot Microtome
Àinventor:: Minot in 1885-
Àinventor 1885-1886
ÀMOST COMMON type used today
especially for paraffin-
paraffin-embedded
tissues.

  
3. Sliding Microtome = MOST
DANGEROUS TYPE DUE TO MOVABLE
EXPOSED KNIFE!
Àinventor/developer:: Adams in 1789
Àinventor/developer
ÀThere are 2 types:
a. Base-
Base-Sledge
> for all forms of media
3
('   
3
('     
3(  
  

  
3. Sliding Microtome
b. Standard Sliding Microtome
 
3
(
3
(
3(   
   

  
4. Rotary Rocking Microtome

5. Vibrotome ± used for unfixed,


unfrozen specimen sectioning for
enzyme demonstrations.
- disadvantage: sections
are liable to disintegrate.

  
6. Ultrathin Microtome ± for cutting
sections for Electron Microscopy
>uses DIAMOND KNIVES
#

-#

 
March 1993:
What is the optimum temperature of
the water bath that is used to float
tissue cut from the microtome?
a. 30 C
b. 37 C
c. 45-
45-50 C
d. 50-
50-56 C
Answer:
³The sections are then floated out on a
water bath set at 45-
45-50 C,
approximately 6-10 C % than the
MP of the wax used for embedding
the tissue.´

Page 107 of the New Gregorios book


%+ ! " ! '!+2
¬ Clearance angle = 0-
0-15 degrees
¬ Bevel angle = 27-
27-32 degrees
¬ The temperature of the hot plate or drying
oven used to dry paraffin sections onto
slides should be at the MP of the paraffin.
¬ For routine work, 76 x 25 mm. slides that
are 1.0-
1.0-1.2 mm. thick are usually
preferred because they do not break
easily.
 
Definition of terms:
1. Chromophores = (Gr. ³color-
³color-
bearers´)
2. Auxochromes = (Gr. ³increasers´)
>when attached to the dye
molecule, they serve to intensify the
color of the dye. They do this by
acting as electron donors to the
chromophore.
3. Lake = the resultant complex of
stain--mordant
stain mordant--tissue.
 
|
    
Hematoxylin ± a natural dye derived
from extraction from the heartwood
of the Mexican tree known as
³Hematoxylin Campechianum´
 
Ripening/Oxidation
>may be done by exposing the substance to air and
sunlight (SLOW)

>may be done by adding oxidizing agents such as:


hydrogen peroxide
mercuric oxide
Potassium permanganate
sodium perborate
sodium iodate
 
I. Alum Hematoxylins
½ Used in routine H and E

½ Produce good nuclear stain (RED)

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)

DESCENDING GRADE OF ALCOHOL

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

Rinse slides in tap water

Acid alcohol (Differentiator)

Ammonia water (Ammonium hydroxide,


lithium carbonate, Scott¶s tap water)
Wash well in running tap water

Stain with Eosin Y

Ascending grade of alcohol

Xylol/xylene

Mount then label


*
¬ Nuclei ± blue
¬ Cartilage and calcium deposits ± dark
blue
¬ Cytoplasm and other tissue

constituents ± varying shades of red


¬ Blood ± bright red
 " +
¬Uses 3 stains: Hematoxylin, OG
OG--6,
EA
Steps:

Fix with 95% ETOH

Stain with Harris Hematoxylin

Acid Alcohol
 " +
Blueing step

Stain with OG-


OG-6 = stains the cytoplasm of mature
(superficial
cells)

70--95% ETOH = for washing


70

Stain with EA 36/50/65 = Stains the cytoplasm of


immature cells (intermediate, parabasal)
 " +
Dehydrate

Xylol

Mount and label


 " +
Eosin azure =

Eosin
Bismarck brown
Lithium carbonate
Phosphotungstic acid
Light green SF
(36,50,65)