UNIT 1: INTRODUCTION TO TISSUE area of abnormality, which you can

PROCESSING now use as a fresh specimen to be

**Specimens are mostly coming from viewed under the microscope

surgery.
SURGERY B. Core needle biopsy
● San galing yung specimen? ● Done in the evaluation of breast
○ Sa surgery masses
○ It doesn’t always have to be ● Compared to the fine needle
coming from the operating aspiration, the needle used here is
room bc we have: large bore, meaning more samples
■ Minor surgery- that you can obtain even small
“glorified clinic” and amounts of the surrounding tissue.
has the materials only ● Once the needle reaches the mass,
for minor surgery the button is pressed, it severes the
■ Major surgery specimen and the surrounding area.
C. Incisional biopsy
FOUR WAYS BY WHICH A SPECIMEN CAN BE ● Gives more amount of specimen
OBTAINED than needle aspiration
A. Fine Needle aspiration ● Require opening up of the area of
● Provide least amount of specimen the lesion.
● Cannot give a diagnosis D. Excisional biopsy
● Using a fine, small bore needle ● Provides most amount of specimen
○ Least invasive ● “Most” meaning:
● Ultrasound Transducer- guides the tip ○ Gives more accurate
of the fine needle to the nodule diagnosis
○ Why so?
■ U need an adequate
Fine needle aspiration sa Thyroid
● The patient is lying down, and you’ll amount of specimen
gonna have a transducer ultrasound to make a diagnosis
here (pointing sa side ng neck) with a ● Require opening up of the area of
monitor in your side, and as soon as you the lesion.
put the needle into the tip of the skin in ● Gives a better accuracy in diagnosis
by the area where you find the nodule
as seen in the monitor, the tip of the
E. Punch biopsy
needle is guided towards the nodule,
you’re actually obtaining from the ● Represents the entire thickness of the
target lesion from the nodule where you skin.
want to take the specimen. Once the ○ Epidermis
tip reaches the target, and aspiration is ○ Dermis
done, you now obtained cells from the ○ Subcutaneous
 ● Contains a cylindrical blade which
cuts into the skin as the device is
rotated while pushing it down.
● Requires anesthesia
F. Shave biopsy
● If a mass is protruding out from the
skin, blade can be used to remove
the mass by scraping it in the level of
the skin to get the protruding lesion
out and make it as a sample.
G. Curettings
● Specifically used for the uterus
● Device called a curet
○ Long handle- introduces the
top of the curet in the uterine
cavity where you will scrape
sample in the endometrium
○ The tip of it has a spoon-like
structure that may have a
sharp edge or a dull edge.
● Scrapings- analyzed
BIOPSY
● Excisional and incisional
AUTOPSY
● Part of forensic pathology
● Purpose is not for diagnosis and
treatment but to determine the
series of events that led to death
COMPARING FRESH TISSUE EXAMINATION
AND PRESERVED TISSUE EXAMINATION
FRESH TISSUE EXAMINATION
● Obtain it and look the tissue under the
microscope immediately
● May be fixed and stained but after it it
goes directly into the slide and look it
under the microscope for examination
● Done for immediate evaluation
● Able to appreciate motion
● Could not be DELAYED (parang regla ko
kasi nakakakaba baka nirape ako ng
mumu)
● If not natignan immediately, it is liable to
develop changes (aw people change
so are tissues) that are commonly
observed when specimens are taken
away from the body as it starts to
deteriorate.
METHODS BY WHICH YOU EXAMINE FRESH
TISSUES
A. Teasing or Dissociation
● You obtain a sample
● Put it on a watch glass with Normal
Saline (NSS) and using a teasing
needle to spread the specimen
away from each other
○ By doing this, you cause the
cellular components to be
farther away from each other
■ Improving the
interpretation as you
will be able to see the
individual cells that
you want to look at
● Teasing- separating cells from one
another (ouch)
B. Squash Preparation
● You have a specimen of 1 millimeter,
out it in the middle of two slides and
forcibly compress it (inipit kalabasa
flavor)
○ Once the specimen is
compressed against each
other, then the specimen is
squashed.
○ You may look at it
immediately after the
microscope or stain the
 slides.
○ By capillary action, the stain
will go into the specimen and
you have a stained
specimen to look at
○ As soon as you obtain,
squash, stain, microscope
● Keyword: FORCIBLY COMPRESS WITH
ANOTHER SLIDE
C. Smear Preparation
● 4 ways of smear prep:
○ Streaking- streak a specimen
onto the slide
■ As the streak goes
distally, adequately
far from each other
■ Better to look at the
distal part of the
streak than the
proximal, since the
proximal part is
thicker
■ Morphology is easily
appreciated in the
distal part
○ Spreading
■ Put the specimen at
the center and simply
spread
■ In spreading, the
sample is usually liquid
■ Must moderately thick
■ Spread away from
the center
○ Pull-apart
■ Specimen on the
center of the slide,
another slide on the
top of the specimen
■ Not compressed and
just pull the slide to
spread the specimen.
○ Touch Preparation
■ Ex: incision made in
the cornea
■ This is only the prep
method that includes
intercellular
relationship
D. Frozen Section
● Physical method of fixation
● Purpose:
○ Rapid diagnosis of tissue
○ Recommended when lipids
and nervous tissue elements
are to be demonstrated
● Method:
○ Thin slices→ 10-15u cut from
fresh tissue frozen on a
microtome with CO2 or on a
cryostat
○ Cryostat- cold chamber (-10
to -20 deg C)
● Applications:
○ Rapid pathologic diagnosis
every surgery
○ Diagnostic and research
enzyme histochemistry
○ Diagnostic and research
demonstration of soluble
substances such as lipids and
carbohydrates
○ Immunofluorescence and
immunohistochemical
staining
○ Some specialized silver stains
in neuropathology
● Rapid freeze- slow freezing causes
distortion due to ice crystal artifacts
○ Liquid nitrogen→ rapid and
commonly availab;e
○ Isopentane cooled by liquid
nitrogen
○ CO2 gas (freezing
microtome)
○ Aerosol sprays (not for muscle
 tissues) ution deg C
● Liquid nitrogen: ● Dissolution of ice crystals
○ Overcools: cracks tissue, ● Proteins are not denatured
biopsy blocks (-70 deg C) ● 4 deg C will complete the
○ Vapor phase forms around fixation process
the tissue causing uneven
cooling making diagnostic CHEMICAL FIXATION
interpretation difficult ● Has 2 mechanisms:
● Isopentane ○ Coagulant fixatives-
○ Liquid at room temp coagulate proteins
○ Prevents vapor phase due to ○ Non-coagulant cross-linking
its high thermal conductivity fixatives- not become part of
the tissue
PRESERVED TISSUE EXAMINATION
● Encompasses all the steps in tissue A. Coagulant fixatives
processing from fixation to labeling ● Coagulate the proteins in the cell
like collagen and lipoproteins
TYPES OF FIXATION ● Not used in ultrastructural analysis
proteins are not denatured.
PHYSICAL METHODS ● For light microscopy
● Two types of coagulant fixative:
A. Heat Fixation ○ Dehydrant coagulant
● Done to accelerate chemical fixatives (not ideal since it
reactions may destroy architecture)
● Ex: Microwave fixation ■ Methyl alcohol
○ Needed to be put in the 100%--> blood smear
fume hood as the fume of and bone marrow
the formalin is harmful when ■ Ethyl alcohol 70-100%
heated. - blood smear,
● If no fume hood available- do not nucleoproteins,
use formalin. enzyme studies
○ Instead, use commercial ■ Alcoholic formalin
glyoxal-based fixatives (Gendre’s)- produce
sputum
B. Freeze-drying and Freeze-substitution ■ Newcomer’s fluid-
● Fix and dehydrate tissues at the nuclear proteins
same time. ■ Carnoy’s fluid-
chromosomes, lymph
Freeze-drying ● Thin sections of specimen glass, urgent biopsies
● Immersed in liquid nitrogen (fixes for 2-3 hrs)
● Vacuum chamber at -40 ○ Acid coagulants:
deg C ■ Bouin’s solution
● Post-fixed ■ Brasil’s alcoholic picro
formol
Freeze-substit ● Immersed in fixatives at -40
D. Non-coagulant cross-linking fixatives
 ● Principle: fixes tissue by forming
proteins
cross-links within & between nucleic
○ Potassium dichromate-
acids between a proteins and
preserves lipids
nucleic acids as well as between
○ Regaud’s (Moller’s)- chromatin,
nuclei and proteins
mitochondria, mitotic figures,
● Two types according to composition
golgi bodies, erythrocytes
○ Simple fixatives
(ultrastructures)
○ Compound fixatives
○ Orth’s- demonstrate bacteria,
in particular your rickettsiae
SIMPLE FIXATIVES ● Lead fixatives - lead acetate
● Formaldehyde- have the tendency to
become a vapor Osmium tetroxide- pale yellow powder that
● Metallic fixatives dissolves in water; strong oxidizing agent
● Osmium tetroxide ● Flemming’s solution- most common
● Flemming’s sol’n without acetic acid-
Formaldehyde: recommended to demonstrate
● 10% solution of formalin mitochondria
● 10% neutral buffered formalin (NBF)-
commonly used COMPOUND FIXATIVES
● pH of 6-8. If low, magkakaroon ng ● Combination of at least two fixatives
artifacts.)
● 10% formol-saline Types According to Action:
● Formol-corrosive- for post mortem ● Microanatomic fixatives- demonstrate
tissues organelles
● Glutaraldehyde- not an irritant. ● Cytological fixatives- cells
○ More expensive but easier on ● Cytoplasmic fixatives- cytoplasm
eyes and mucus membranes ● Histochemical fixatives- histochemistry
○ More pleasant than
formaldehyde
○ 2 formaldehyde residues linked
by 3 carbon chains

Metallic fixatives:
● Mercuric chloride
○ Zenker’s fluid- for small pieces
of liver, spleen, connective
tissue, nuclei
○ Zenker’s formol (helly’s)- for
pituitary gland, bone marrow,
and blood containing organs
○ Heidenhan’s Susa-for tumor
biopsies of the skin
● Chromate fixatives
○ Chromic acid- precipitate all