Histopath module 1 Flashcards
may vary according to: the structural
and chemical components of the cells to be examined or studied, the
nature and amount of the tissue to be evaluated, and the need for an
immediate examination of a tissue structure.
FRESH TISSUE EXAMINATION
process whereby selected tissue specimen is immersed in a watch
glass containing isotonic salt solution, carefully dissected or
separated, and examined under the microscope. Either unstained by
phase contrast microscope or bright field microscope or stained with
differential dyes.
Teasing or dissociation
is a process whereby small pieces of tissue not more than one mm. in
diameter are placed in a slide and forcibly compressed with another
slide or with a cover glass
SQUASH PREPARATION
process of examining sections or sediments, whereby cellular
materials are spread lightly over a slide means of a wire loop. This
technique is especially usefully in cytological examinations,
particularly for a cancer diagnosis.
SMEAR PREPARATION
THREE WAYS OF SMEAR PREPARATION
- STREAKING
- SPREADING
- PULL APART
with an applicator stick, the material is rapidly and gently applied in
direct or zigzag line throughout the slide
STREAKING
has the advantage of maintaining a cellular inter relationships of the
material to be examined. It is especially recommended for smear
preparations of fresh sputum and bronchial aspirates and also for
thick mucoid secretions.
SPREADING
- useful for preparing smears of thick secretions such as serous fluids,
concentrated sputum and blood smears.
PULL APART
- for rapid diagnosis, especially recommended when lipids and nervous
tissue elements are to be a demonstrated
FROZEN SECTIONS
COMMONLY USED METHOD IN FREEZING TISSUE
Liquid nitrogen
* Isopentane cooled by nitrogen
* Carbon dioxide gas
* Aerosol sprays
10 STEPS IN PROCESSING TISSUE
- FIXATION
- DEHYDRATION
- CLEARING
- INFILTRATION (IMPREGNATTION)
- EMBEDDING
- TRIMMING
- SECTION-CUTTING
- MOUNTING
- STAINING
- LABELLING
First and most critical step in histo technology involves fixing or
preserving fresh tissue for examination.
FIXATION
to preserve morphologic and chemical integrity of the
cell in as life like a manner as possible.
PRIMARY AIM
: harden and protect the tissue from the trauma of
further handling.
SECONDARY GOAL
It is also prevents degradation decompositions , putrefaction and
distortion of tissue after removal from the body.
SECONDARY GOAL
TWO BASIC MECHANISM INVOLVED IN FIXATION
ADDITIVE FIXATION? NON ADDITIVE
fixatives is taken in and becomes part of the tissue by forming cross- links or molecular complexes and gibing stability to the protein
. ADDITIVE FIXATION
whereby the fixing agent is not incorporated into the tissue, but
ALTERS the tissue composition and stabilizes the tissue by removing the
bound water attached to H-bonds of certain groups within protein
molecule
. NON ADDITIVE FIXATION
MAIN FACTORS INVOLVED IN FIXATION
Hydrogen Ion concentration
temperature
thickness of sectioN
osmolalitY
concentrations
duration of fixations
Hydrogen Ion concentration ph?
ph must be 6 -8
temperature
for surgical specimens?
room temp
For elctron microscopy and histochemsitry
0-4 deg cel
osmolality
should be slightly hypertonic solutions
400-459 mOsm. Isotonic are 340 mOsm
PRACTICAL CONSIDERATIONS OF FIXATIONS
SPEED
PENETRATION
VOLUME
duration of fixation
-to prevent autolysis and putrefaction.
SPEED
10-25 X the volume of tissue. Maximum effective is 20x
VOLUME
CHARACTERISTICS OF A GOOD FIXATIVE
- Must be a cheap
- must be stable
3.must be a safe to handle - must kill the cell quickly thereby producing minimum distortion of
cell constituents - must inhibit bacterial decomposition and autolysis
TYPES OF FIXATIVES ACCORDING TO
COMPOSITION
SIMPLE FIXATIES
METALLIC FIXATIVE
COMPOUND FIXATIVE
TYPES OF FIXATIVES ACCORDING TO
COMPOSITION
SIMPLE FIXATIES example: 1-2
A. formaldehyde
B. glutaraldehyde
TYPES OF FIXATIVES ACCORDING TO
COMPOSITION
Mettalic Fixatives
A.mercuric chloride
B.chromate fixative
C. lead fixative
D. heat
TYPES OF FIXATIVES ACCORDING TO
COMPOSITION
METALLIC FIXATIVES
Under B.chromate fixative 1-2
- potassium dichromate
- chromic acid
TYPES OF FIXATIVES ACCORDING TO
COMPOSITION
Under ng C. lead fixative 1-5
METALLIC FIXATIVES
picirc acid
acetic acid
acetone
alcohol
osmium tetroxide
TYPES OF FIXATIVES ACCORDING
TO ACTION
A. MICRONATOMICAL FIXATIVES
B. CYTOLOGICAL FIXATIVE
TYPES OF FIXATIVES ACCORDING
TO ACTION
-Those permit the general microscopic study of tissue structures without
altering the structural pattern .
MICRONATOMICAL FIXATIVES
ACCORDING TO ACTION
A. MICRONATOMICAL FIXATIVES EXAMPLE
1-8
10% formol saline
* 10% neutral buffered formalin
* heidanhain’s susa
* Formol sublimate
* zenker’s sol
* zenker- formol
* bouins sol
* Brasil’so
ACCORDING TO ACTION
B. CYTOLOGICAL FIXATIVES
Nuclear fixatives example 1-5
Nuclear fixatives
1.1 flemming’s fluid
1.2Carnoy’s fluid
1.3Bouin’s fluid
1.4Heidenhain’s susA
B. CYTOLOGICAL FIXATIVES
- they have ph of 4.6 or less
Nuclear fixatives
- CYTOPLASMIC FIXATIVE
2.1 aCETIC ACID
2.2 flemmings fluid w/o acetic acid
2.3 kelly’s fluid
2.4 Formalin w/o post chroming
2.5 Orths Fluid2.1ACETIC ACID
3.HISTOCHEMICAL FIXATIVE
3.1Formol saline 10%
3.2Absolute ethyl alcohol
3.3Acetone
3.4Newcomer’s fluid
-fixes but does not precipitate cytoplasmic structures.
-preserves lipids and mitochondria
POTASSIUM DICHROMATE
- recommended for demonstration of golgi bodies.
- Does not preserve fats
REGARD’S (MULLER’S) FLUID
- recommended for study early of degenerative processes and tissue
necrosis. - Demonstrates rickettsiae and other bacteria.
ORTH’S FLUID
- recommended for acid mucopolysaccharides
- Fix connective tissue mucin
LEAD FIXATIVE
- excellent fixative for glycogen demonstration.
- yellow color may produced, it can be removed treatment with another
dye (lithium carbonate)
PICRIC ACID
- recommended for fixation of embryos and pituitary biopsies.
- does not need washing out.
. BOUIN’S SOLUTION
- excellent fixative for glycogen.
BRASILS ALCOHOLIC PICROFORMOL FIXATIVE
- fixes and precipitates nucleoproteins
- solidify at 17 deg cel
GLACIAL ACETIC ACID
- used as both fixative and dehydrating agent
ALCOHOL FIXATIVES
- excellent for fixing dry and wet smears, blood smear and bone
marrow smear.
METHYL ALCOHOL
- recommended for fixing chromosomes, lymph glands and urgent
biopsies. - most rapid fixative
CARNOY’S FLUID
- most common chrome osmium acetic acid fixative
- recommended for nuclear preparation
FLEMMING’S SOL
-RECOMMENDED FOR CYTOPASMIC STRUCTURE PATICULARY
MITOCHONDRIA
FLEMMING’S SOL WITHOUT ACETIC ACI
-Use as weak decalcifying agen
TRICHLOROACETIC ACID
recommended for the study of water diffusible enzymes esp.
phosphatase and lipase. fixing brains and Diagnosing rabies
ACETONE
- to facilitate and improve demonstration
- to make special stain possible
SECONDARY FIXATION
DECALCIFICA TION
* Calcium may be removed by the following
acid
Chelating agents
Ion exchange resins
electrical ionization
- Most common and the fastest decalcifying agent used so far. However
it inhibits nuclear stain and destroying tissues especially in
concentrated solutions.
NITRIC ACID
- Recommended for urgent biopsies and for needle and small biopsy
specimens to permit rapid diagnosis within 24 hrs. or less.
Aqueous nitric add solution 10%
- Rapid acting; recommended for urgent biopsies
- Produce yellow color; remedy: neutralizing tissue in 5% sodium
sulfate and washing in running tap water for atleast 12 hrs. addition
of 0.1% urea to pure concentrated nitric acid will also make
discoloration disappear without affecting the efficiency of the
decalcifying agent.
FORMOL-NITRIC ACID
- Recommended for routine purposes
- Nuclear and cytoplasmic staining is good.
- Maceration is avoided due to the presence of chromic acid and
alcohol - Not recommended for urgent work.
- Composed of chromic acid, ethyl alcohol and nitric ac
PERENYI’S FLUID
- Most rapid decalcifying agent
PHLOROGLUCIN NITRIC ACID
- Moderately rapid decalcifying agent.
Does not require washing out
before dehydration.
Recommended for teeth and small pieces of bone
VON EBNER’S FLUID
- Process by which processed tissue, most commonly paraffin
embedded tissue, is trimmed and cut into uniformly thin slices or
sections to facilitate studies under he microscope. And that is
microtomy.
MICROTOMY
THREE ESSENTIAL PART
- BLOCK HOLDER
- KNIFE CARRIER AND KNIFE
- PAWL, RATCHER FEED WHEEL AND ADJUSTMENT SCREWS
- For cutting serial sections of large blocks of paraffin embedded
tissues. - It is invented by Paldwell Trefall in 1881.
- It is considered as simplest type of microtome.
- It is not recommended for serial sections since tissues are cut in
slightly curved planes.
ROCKING (CAMBRIGE MICROTOME)
- for cutting paraffin embedded sections.
- It is invented by Minot in 1885-1886.
- This is the most common type used for both routine and research
laboratories.
ROTARY (MINOT) MICROTOME
- Used for cutting celloidin embedded sections.
- It was developed by Adams in1789.
SLIDING MICROTOME
