5 - Introduction To Tissue Processing

HPCT 311: Histopathologic/Cytologic Techniques│Lecture
2022-2023 3RD YEAR, 2ND SEMESTER OUR LADY OF FATIMA UNIVERSITY | COLLEGE OF MEDICAL LABORATORY SCIENCE
o curette – a surgical instrument that has a

Introduction to Tissue scoop, ring, or loop at the tip
Processing
(Midterm, 1st Topic)
Trans Outline:
I: Tissue Processing Processing
II. Surgical Procedures V: Factors of Tissue
III: Specimen Received Processing
for Processing VI: Technical
A. Methods of Fresh Considerations
Tissue Exam VII: Frozen Section
IV: Conventional Tissue
TISSUE PROCESSING
 A better and more effective means of studying the  Excision Biopsy

tissues, whether normal or abnormal, is by o removes the entire area in question
examination of their sections and smears which have o aka excisional biopsy
been permanently preserved, stained, and mounted on
glass slides with cover slips on top for permanent
keeping.
 This overall process is called tissue processing
SURGICAL PROCEDURES TO OBTAIN SPECIFIC

TYPES OF TISSUE
 Core Needle Biopsy

o removes not only cells but also small
amount of surrounding tissue
o provides additional information to assist in
the examination of lesion
o aka Incision Biopsy because in a way, these
two are somewhat getting parts of what we  Fine Needle Biopsy (FNAB)
are after o uses smallest needle to remove cells from
the area in question
o not always adequate to obtain a diagnosis,
depending on the area to be biopsied
o simplest, least invasive test
 Curetting
o tissue that is scooped or spooned to remove
tissue or growths from body cavity such as
endometrium or cervical canal
o needed in a curettage biopsy, which can be
done on the surface of tumors or on small
epidermal lesions with minimal to no
topical anesthesia using a round curette  Incisional Biopsy
blade o only a portion of tissue is removed
1
HPCT 311 | BSMLS 2024 HISTOPATHOLOGIC/CYTOLOGIC TECHNIQUES STUDENT NOTES ONLY| PREPARED BY: CRUZ, A.M.A.
o if found cancerous, further surgery may be

needed to remove or excise the entire lesion
o aka core biopsy
o takes out even more surrounding tissue
o takes out some of the abnormality but not
all
o the doctor will slice into the lesion and
remove only a portion
 Shave Biopsy
o small fragments of tissue are “shaved” from
the surface
o done with either a small scalpel blade or
curved razor blade
o usually done on skin
SPECIMEN RECEIVED FOR PROCESSING
 Preserved
o Advantage: permanent
o use of a fixative (chemical or reagent)
usually formalin
o When sample is received in a formalin,
there is no need to reject. Proceed to a
routine surgical procedure and check the
volume of formalin included
 Punch Biopsy o ideally, specimen should have at least 20
o primary technique for obtaining diagnostic
times of volume for formalin
full- thickness skin specimen  Fresh
o requires basic general surgical and suture-
o Advantage: examined in living state
tying skills  still allow protoplasmic activities
o use of circular blade that is rotated from
 can see the motion (mitosis and
epidermis down to subcutaneous fat phagocytosis)
yielding 3-4 mm cylindrical core of tissue
o Disadvantage:
sample  not permanent
 epidermis  dermis 
 develop changes observed after
subcutaneous fat death
o usually easy to learn
o no addition of any reagent or chemical
o circular blade ranges 1-8 mm which is
o encountered in the following:
attached to a pencil-like handle
 for frozen sections – tissue is
received as fresh for immediate
2
microscopic evaluation by  Squash Preparation

pathologist o aka Crush Preparation
 for operating room (OR) o squashing / crushing
consultation o small pieces of tissue (not more than 1 mm
- pathologist will look at in diameter) is placed in a slide and
specimen and make a compressed by another slide/ cover glass
gross diagnosis o visualization:
- assistant can weigh and  supravital stain placed at the
measure specimen, make junction of slide and cover glass
markings or ink the absorbed by tissue through
specimen if necessary capillary attraction
METHODS OF FRESH TISSUE EXAMINATION:
 Teasing and Dissociation

o tissue specimen is immersed in isotonic
solution in petri dish or watch glass
 normal saline solution (NSS)
 Ringer’s solution / Ringer’s
lactate solution / Sodium lactate
solution / Lactated Ringer’s /  Smear Preparation
Hartmann’s solution o differs depending on material to be
- mixture of sodium examined
chloride, sodium lactate,  examining the sections or
potassium chloride, and sediments whereby the cellular
calcium chloride in water materials are spread lightly over
designed to match the the slide by using an applicator
composition of plasma stick, platinum wire loop, or
o needle: dissect tissue appositional second slide
o applicator stick: separate tissue with direct
o general rule: by a platinum loop or by a
or zigzag spread
second slide
o visualization:
o examination: fresh or by supravital stain
 wet preparation with supravital
 can be made permanent by fixing
stain or differential dyes
tissue while still wet, stain and
 wet preparation unstained and use
mount
Phase contrast microscope or
Brightfield microscope - useful for thick
secretions (serous fluids,
- selected pieces of a tissue
sputum, enzymatic
are transferred carefully lavage from GIT, bone
to a microscope slide and marrow preparations,
mounted as a wet and peripheral blood
preparation underneath smears)
a cover glass (make sure o useful for cytological examinations (cancer
to avoid bubbles) diagnosis)
o advantage:
 cells examined in living state Methods:
o disadvantage: - Streaking
 not permanent o applicator stick or platinum loop: rapidly
and gently spread by direct or zigzag line in
slide
o too thick or thin: make tissues unsuitable
for examination
o there should be uniform distribution of
secretion
3
 Touch Preparation
o aka Impression Smear
o leaving an impression on the surface of the
slide
o specialized method
o surface of freshly cut piece of tissue is
brought into contact and pressed on to the
- Spreading surface of clean slide allowing the cells to
o selected portion of material is transferred to be transferred directly to the slide for
a clean slide examination by phase contrast
o spread into a moderately thick film: teasing o visualization:
mucous strands apart with applicator stick  unstained for phase contrast
o Advantage: maintain the cellular microscope
interrelationship  stained for bright field microscope
o Disadvantage: tedious than streaking o Advantage: maintain cellular
o Recommended for: fresh sputum, bronchial interrelationship
aspirates, other thick mucoid secretions
CONVENTIONAL TISSUE PROCESSING
 Steps are required to take tissue from fixation to a state

where it is completely infiltrated with suitable waxes.
Later on it can be embedded ready for section cutting
- Pull-apart in the microtome.
o Placing drop of secretion or sediment upon  The tissues (solid structures), can be preserved and
one slide and facing it to another clean must be preserved and carefully processed in the
slide. Upon doing that, the material following order: (1) Numbering (2) Fixation (3)
disperses evenly over the surface of the two Decalcification (4) Dehydration (5) Clearing (6)
slides Impregnation (7) Embedding (8) Trimming (9)
o slight movement of two slides in opposite Section-cutting (10) Staining (11) Mounting (12)
directions may be necessary to initiate the Labelling
flow of materials. Afterwards, two slides
will be pulled apart in a single,  Numbering
uninterrupted motion o complete request form
o immediate examination necessary with or  lists the patient information,
without vital stain history and site of origin for the
tissue submitted
 reject if:
- unlabeled specimen
- incomplete information
in requisition form
- incomplete specimen
based on request form
o tissue accessioning
 C: cytology specimen
 A: autopsy specimen
4
 S: surgical specimen  Dehydration

o example: S-19-0001 o process in which the water content in the
 S – surgical tissue to be processed is completely
 19 – for its year reduced
 0001 – serial number which o by passing the tissue through increasing
depends on the rules of laboratory concentration of dehydrating agents
(eg: this is the first specimen for o since later, tissue will be subjected to a
2019) melted paraffin which is hydrophobic
o each specimen receives a certain accession (immiscible with water) so most of the
number water in specimen should be removed
o each number is unique to particular case before infiltrated with wax
and never reused o agents:
o case / accession number should be placed  ethyl alcohol – poor fat solvent
in specimen container, requisition slip - 70%: 15 mins
written on upper right corner, and it should - 90%: 15 mins
be the label of tissue cassettes instead of - 100%: 15 mins
patient’s name - 100%: 15 mins
 Fixation
- 100%: 30 mins
o tissues should be fixed as soon as possible
after arriving in the laboratory placing the - 100%: 45 mins
tissue in a liquid fixing agent (fixative) - increasing concentration
 formaldehyde/ formalin is done to avoid excessive
o purpose: preserve tissue permanently in a distortion of tissue until
life-like state as possible water-free tissue is
o fixative should be 10-20x more in terms of reached
volume than the specimen - water soluble proteins
o if fixed already upon receipt, ask how long are removed at lower
it has been submerged concentrations of ethanol
- when ethanol reaches
100%, there are still
certain lipids that may be
dissolved
 Acetone / Isopropyl alcohol
- to ensure complete
dehydration, some make
use of a superior fat
solvent
 Decalcification - should be added before
o process of removal of the calcium salts the final absolute ethanol
from the specimen  chloroform or trichloroethane:
o agents: transition solvent
 nitric acid - added after using
 hydrochloric acid superior fat solvent and
 formic acid
before proceeding with
 picric acid
 acetic acid final concentration
 citric acid  Clearing
o optional depending on specimen o alcohol in the tissue is replaced by fluid
which will dissolve the wax used for
impregnating the tissue
o also removes fat from tissue which may be
a barrier to wax infiltration
o agents:
 xylene : most common since it is
miscible with both ethanol and
paraffin
5
Xylene: 20mins
- another molten wax to form solid tissue
Xylene: 20mins
- block. This can later be clumped into
Xylene: 45mins
- microtome for sectioning.
 cedarwood oil: best agent but
expensive
 benzene: carcinogenic
 chloroform: toxic and expensive
o not just to clear remaining alcohol as it can
impart optical clarity or transparency to
tissues due to their relatively high
refractive index
o the dehydrated tissue is transferred to
solvent
o solvent for clearing should be miscible with
both remaining alcohol and paraffin wax
o following dehydration, tissue is immersed
in 1-3 different xylene immersions wherein
ethanol is gradually replaced with xylene
o Note: when tissue is later on embedded,
xylene will be replaced by molten wax
 Impregnation
o tissue is kept in a wax bath containing
molten paraffin
 Paraffin wax: 30 mins
 this example sequence is also for
tissue sections not more than 4 mm
thick
o Paraffin wax + additives (resins such as
styrene or polyethylene) will allow the thin
sectioning using microtome
o increases optical differentiation and
hardens the tissue which makes section
cutting easier
o infiltration  Trimming
o paraffin wax is liquid at 60℃ and solidifies  Section-cutting
at 20℃ o blocks are cut or sectioned and thin strips of
o it has to solidify since it will be later on cut varying thickness are prepared
to different sections o use of microtome
o result should form ribbons that can flatten o solid block can now be clumped on
fully when float in warm water bath microtome for section cutting
 Embedding o should be thin enough to be placed on slides
o transferring the tissue to a mold filed with o one of the most directly correlated factor is
molten wax and is allowed to cool and the thickness in which the specimen is cut
solidify o thickness
o most important step in embedding: correct  specimens for routine hematoxylin
orientation of block in mold and eosin staining – cut in 3-5 μm
o after infiltration with liquid state, it is thickness
allowed to cool down and solidify in  for amyloid deposits – sectioned at
embedding stage 8-12 μm
o tissue is oriented and placed inside the  kidney biopsies – cut at 2 μm for
embedding mold optimal doing of glomeruli
o After proper orientation, half of the paraffin structures
is placed and allowed to solidify then tissue
is placed on top and filled again with
6
o must be placed in floatation bath so that it there is no need to remount preparations

will not crease as it should be straight and for microtomy
flatten out on the slide  Staining
o done to bring out the particular details of
the tissue under study
o haematoxylin and eosin stain: most
common
o to put a contrast since if unstained, it will
look nearly invisible
o After staining, can still proceed with
viewing immediately under microscope but
there will be interferences like differences
in light refraction between glass slide and
tissue component that can lead to
differences in the length of dispersion, so
very little microscopic details can be
appreciated.
o Floatation Bath
 sections “floated out” on the
 Mounting
surface of warm water in a
o Subject or impregnate tissue with a
flotation bath to flatten them and
transparent medium that has a refractive
then picked up onto microscope
index close to the glass and tissue –
slides
mounting medium
 After thorough drying they are
 syrupy fluid / viscous
ready for staining
 applied between the section and
cover slip after staining
 sets the section firmly, preventing
the movement of coverslip
 protects the stain section for
getting scratched
 facilitates easy handling and
storage of slides
 prevents bleaching or
deterioration of structures due to
oxidation thereby it can also
preserve the slides for permanent
keeping
 can help prevent the distortion of
image during microscopic
examination
o adhesives used for fixing the sections on the
o It is advisable to save any unmounted slides:
paraffin ribbons with appropriate  albumin solution (Mayer’s egg
identification because in such cases they albumin)
can be reaccessioned after a week or two so  starch paste
7
 gelatin oHigh temperature: causes tissue to shrink

and become brittle, hard
o Low temperature: increases viscosity of
reagents, causing reduced rate of diffusion
while increasing the processing time
o maintain embedding waxes 2-3℃ above
melting point
 Vacuum and Pressure
o reduced pressure: increases infiltration rate
and decreases processing time
o high pressure: facilitates infiltration of
dense specimens with more viscous
embedding media
o vacuum application during infiltration:
improve processing quality and aid in
removing the trapped air from porous
tissue
o reduce infiltration time when dealing with
fatty and dense tissue
o vacuum application during dehydration,
clearing and infiltration: improve the
 Labelling quality of processing in the tissues such as
lungs which becomes de-aerated during the
FACTORS AFFECTING DURATION OF TISSUE process
PROCESSING AND EXTENT OF FILTRATION o duration of infiltration is independent of
vacuum; still dependent on viscosity of wax
 Tissue density and thickness
o spongy tissues (lungs) TECHNICAL CONSIDERATIONS
 spongy – rapidly infiltrated than
that of dense tissue due to their  baskets & metal cassettes should be clean and wax-
spaces free
o thickness of tissue  tissues should not be packed too tightly in baskets
 also affects the rate of reagent  fluid levels must be higher than the specimen
diffusion and time of processing containers
 thicker = longer time  timing and delay mechanism must be correctly set and
 Agitation checked against the appropriate processing schedule
o increases flow of fresh fluids in or around  gentle washing and minimal thickness of cell layers
the tissues will prevent cells from detaching during staining
o fluid interchange between processing  quality of staining can be compromised by inadequate
reagents and tissues is promoted by fixation and similarly by poor tissue processing
exposure of the maximum tissue surface  make sure glass slides are clean, scratch-free, free
area from debris
o tissue in cassettes  needle biopsies and bloody specimens: incubated
 has grills which are spaces where conservatively
agents can enter  fatty specimens: processed longer than usual/average
 tissues inside should be loosely
packed to facilitate exchange of FROZEN SECTION
reagents and increase diffusion
o without agitation, tissues tend to settle to  to see rapid diagnosis of a pathologic process
the bottom of processing device or can  Vacuum use of microtome with CO2 or cold chamber
become too tightly packed, reducing the (cryostat) kept at an atmospheric temp of -10 to -20℃
surface area available for fluid exchange  recommended for demonstration of lipids and nervous
 Temperature tissue
o 37-45℃ for limited time can speed up fluid  also used for muscle and nerve biopsies
penetration and tissue processing protocols  uses:
o rapid pathologic diagnosis during surgery
8
o used for intra-operative pathology to aid  when small crystals start forming,
surgeons in decision for next plan of action suggest that already in
o diagnostic and research enzyme approximately -170℃
histochemistry o tissue to be frozen is affixed in cork disc,
o diagnostic and research demonstration of aluminum foil, or cryostat chuck and dropped
soluble substances (lipids & CHO) into cooled liquid isopentane
o immunofluorescent and o excellent for freezing muscle tissue
immunohistochemical staining  CO2 GAS
o specialized silver stains in neuropathology o conventional
 Advantages: o CO2 gas from CO2 cylinder
o rapid processing time  AEROSOL SPRAY
o less equipment requirement o for small pieces of tissue except muscle tissue
o less need for ventilation o quick-freezing spray cans of fluorinated
 Disadvantage: hydrocarbons such as cryokwik which has a
o poor quality of final slide since preparations distinct advantage of rapidly freezing blocks
are rapid of any type of tissue
 slow freezing: distortion of tissue due to ice crystal  Fresh, completely unfixed tissues or tissues that have
artifacts been briefly treated with formalin
o may not require embedding
METHODS OF FREEZING o frozen and cut in freezing microtome or
cryostat
 LIQUID NITROGEN
o methods of preparing frozen sections:
o used in histochemistry and during
 Cold knife procedure
intraoperative procedures
 Cryostat procedure – using cold
o Advantage: most rapid
microtome
o Disadvantage:
 soft tissue is liable to crack due to REFERENCES
the rapid expansion of the ice within
the tissue producing ice crystal o Gregorios-Bruce, Jocelyn (2012).
artifacts Histopathologic Techniques 2nd ed. Quezon
 overcools urgent blocks---damage City: JMC Press.
both block & blade if sectioning is
done at -70℃ or below: temperature
equilibrium (cryostat chamber
should be in equilibrium first with
blade and samples to be used before
sectioning)
 majority of non-fatty unfixed tissue:
sectioned between -10 to -25 ℃
 formation of vapor phase around the
tissue acting as insulator----uneven
cooling of tissue----difficult
diagnostic interpretation
 can be overcome with the
use of other reagent
(Isopentane or Freon 2.2 –
both has a high thermal
conductivity)
 ISOPENTANE COOLED WITH LIQUID
NITROGEN
o liquid at RT
o Pyrex glass beaker containing isopentane is
suspended in a flask of liquid nitrogen until
half liquid and half solid stage is reached
o beaker is removed when small crystals start
forming on the side of beaker
9

5 - Introduction To Tissue Processing

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5 - Introduction To Tissue Processing

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HPCT 311: Histopathologic/Cytologic Techniques│Lecture

o curette – a surgical instrument that has a

 A better and more effective means of studying the  Excision Biopsy

SURGICAL PROCEDURES TO OBTAIN SPECIFIC

 Core Needle Biopsy

o if found cancerous, further surgery may be

SPECIMEN RECEIVED FOR PROCESSING

microscopic evaluation by  Squash Preparation

METHODS OF FRESH TISSUE EXAMINATION:

 Teasing and Dissociation

CONVENTIONAL TISSUE PROCESSING

 Steps are required to take tissue from fixation to a state

 S: surgical specimen  Dehydration

o must be placed in floatation bath so that it there is no need to remount preparations

 gelatin oHigh temperature: causes tissue to shrink

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