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PART FIVE (5)
Histotehnology and Histopathology
Clinical Laboratory Diagnosis
For
General Laboratory Scientists
Histotechnology and Histology
By
Kourosh Teymourian, CLS
561
Contents
Pages
Chapter One (1)
1.
2.
3.
4.
General Concepts
560
Instrumentation in Histopathology today
561
Safety in Histotechnology
565
Collection and reception of specimen and specimen process in front end aspect569
Chapter Two (2)
1.
2.
3.
4.
5.
6.
7.
560
Fixation and fixatives used in Histotechnology
Application of microwave and tissue processing
Microscopy
Staining principles
Stains
Tissue staining
Hematoxylun and Eosin
571
571
576
581
583
584
586
587
562
Chapter Three (3)
1.
2.
3.
4.
5.
589
Staining of mucins
Staining of connective tissue
Staining of pigments
Bone processing and staining
Amyloid substance staining
589
591
599
608
614
Chapter Four (4)
1.
2.
3.
4.
5.
6.
616
Histopathology techniques in neurology (staining properties)
Neuroendocrine staining and related stains
Routine immunohistologic stains in Histopathology
Immunohistochemistry of Lymphomas
Immunohistochemistry of Breast pathology
Histochemistry by enzyme applications
616
620
624
630
634
637
References
641
Normal Values
648
Chapter One (1)
General Concepts
H
istotechnology is a branch of
medical
science
particularly
surgical pathology that deals with
preparation,
processing
and
staining of tissues for the tissue evaluation
and interpretation of the diseased tissue(s)
and organs involve in tissue pathology. The
result is a tissue slide or film that will be
studied microscopically or in some cases
by automated tissue sorter and result will be
registered by the pathologist or his
assistant/designate.
Pathology is the science of studying the
disease process: it is divided into two main
branches, 1) Clinical pathology which
deals with detection of disease by means of
diagnostic reagents, antisera and other
chemical and serological means, this is
under Canadian or American laboratory
medicine program, and second: 2) is
anatomical or surgical pathology which
deals
with
tissues (histology and
histopathology), cells in the fluids (such as
cytology and cytopathology) and processes.
These biopsied or autopsied tissue sections
are processed to identify and determine
disease status and its implication on the
patient. Another closely related branch of
medical science under surgical pathology is
the
cytotechnology and cytology
(cytpopathology): a division that uses
secretion and body fluid cytology to detect
561
and study disease marker in the cells of the
body fluids and the cells’ exfoliated layers
to detect cancerous tissues or other diseases.
Histology is the study of normal tissues so
that by means of histotechnology and its
application we can prepare tissue slides to
interpret the tissue status under study by the
pathologist. It is essential for the laboratory
scientists
or
pathology
assistants/histotechnologist/technician
to
have thorough knowledge of normal tissues
histology and the principles of tissue
processing and staining. In this connection
histopathology is the study of disease and
affected tissues by mean of tissue sections
prepared by histotechnologic applications.
The surgeon or the physician may send
tissues/organ(s) incised or excised along
with tissue biopsies, autopsies and fine
needle aspirations (biopsies) to surgical
pathology department to diagnose and
determine the cause of tissue damage or
illness by tissue microscopy evaluation.
Histotechnology (& histology) and its
application
is
the
domain
of
histotechnologist. This branch of
medicine is the concern of this section/part
five of this textbook.
Quality Management (QM) and Quality
Assessment/Assurance (QA) similar to
other departments in laboratory medicine
department need training of staff, medical
laboratory team and other relevant
personnel. Quality management embraces
aspects such as Quality Assurance
(assessment) system and scheme, which is
the assessment process and monitoring
quality control and assurance processes.
Quality Control although are parts of all
clinical laboratory departments it is not
excluded in surgical pathology lab(s) as it
maintains Standard Operation Procedure
(SOP)/(Kennedy A et al.).
Quality assurance process should include
standard preparation of solutions and
stains. Special stain preparation for
immunohistochemistry,
chemical
preparation, turnaround time for routine and
special stains, such as hematoxylin, eosin
and Carmine’s etc must follow the
assessment
and
laboratory
Quality
Assurance expectations. Staining procedures
and chemical preparations for making the
required staining solutions play a crucial
role in final interpretation of the stained
tissue section of the slide and must be
considered as a Quality Assurance tool. Still
for a better Quality Assessment/Assurance
proper functioning and maintenance of
tissue
processors,
TissueTek®
systems and routine inspections of
instruments and equipment maintenance are
a must and should follow according to the
standards and guidelines of CLSI (Clinical
and Laboratory Standard Institute) and CAP
or CRCP [American college of
pathologists and Canadian College of
Pathologists].
Quality Assurance scheme for stain
preparation and quality may be done by
checking the archival section in the
department or participating in an external
quality control scheme/program/audit or
using the schedules of ISO 9000
international Quality Control standards.
These are for establishing a better quality
management system. In this regards CLSI
(Clinical and Laboratory Standard Institute)
previous NCCLS (National Committee on
Laboratory Standards) can be a source of
data acquisition for the quality management,
and assurance purposes. Histotechnologists
scientists/technologists and technicians must
be accredited/member by/of CMLTO
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(College
of Medical Laboratory
Technology of Ontario), CSMLS or
OSMT (Canadian Society for Medical
Laboratory Science & Ontario Society for
Medical Technologists) or by other US
regulatory bodies (ASCP, American College
of Clinical Pathologists, and National
Certification Agency/NCA & CNC)/ (Paget
G E et al.).
Instrumentation
Histopathology Lab
in
(Kennedy A et
al)
Today histopathology laboratory is equipped
with many sophisticated instruments and
equipments
such
as
TissueTek®
embedding
system(s),
tissue
processors, tissue sorter, computer attaché
to the system equipment, microtome(s), and
cryostat, microscopes ( & stereomicroscope)
and even electronic microscope for research
purposes, tissue floatation water-bath, slide
warmer, paraffin wax trimmer, convection
ovens and microwave heater. Although in
large, in some laboratories, most of the
processes are still done manually such as
preparing solutions for stains, staining
solution changes, reception of the
specimen,
processing
of
specimen,
interpretation and reporting of results. The
idea of a total automation in this part of
clinical laboratory is not universally
crystalized. The area of specimen reception
uses skilled technicians to handle the
primary aspect of correct reception and
identification of the specimen and labeling
of slides and the processing of tissues by a
histotechnologist/assistant pathologist, still
seems to be not fully automated and is
done by these personnel. The interpretation
of results which mandates a pathologist is
still the same as before. However these
processing lines in some advanced surgical
pathology lab have fairly fulfilled this
promise to some extends in the pathology
departments.
Instruments as tissue processors are of two
kinds the one with old rotary system used
before 1980s and the new type of tissue
which is manufactured and utilized after
1980s: it is known as “fluid change
systems”. In the rotary system or carousel
type, the pumping fluid is done through each
changing cycles for tissue-cassettes in each
bucket/basket/chamber
with
pumping
tubes/pipes supplied for each cycles and the
basket rotates to reach the system pumps
at different stages. Whereas in the fixed
pumping fluid system (fluid exchange)
handles and processes tissue cassettes in one
single bucket/basket with all reagents
pumped into that the same basket with
exchanging fluids. In the latter system the
fixing solutions, dehydrants, clearing
agents and infiltrating medium are
exchanged in the same basket, nevertheless
in former system basket moves to different
stages or stepping cycles to have the said
processing solutions. The following figure
(Fig. 1-1) is the new fluid exchange
system type from KD-TS3D: KEDEE
system®, automated tissue processor shown,
other as technicon processor® by Technicon
incorporation are also marketed in the US,
Canada and elsewhere.
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the tissue holding cassettes for embedding
and a TissueTek system for wax
dispensing.
Fig. 1-1: Fluid exchange automatic tissue
processor, KD-TS3D® courtesy of KEDEE
systems.
In fluid exchange system, once the fluid
is in the system/chamber, penetration is
aided by means of various methods such as
vacuum-pressure,
vacuum
only
or
alternating cycles, etc. unique to the
manufacturers. Once the samples are fed
into the chamber the lid is airtight and
process commences. At the last sequence
which is the evacuation of paraffin wax
from the chamber: the system must be
rinsed with cleaning solution to avoid build
ups of the paraffin wax which is
incompatible with the fixing solution at the
next run/stage. The processor is capable of
alarming and alerting the staff of the
situation which therefore may prevent the
hanging and drying of the tissue blocks
during a black-out or burnout.
(A)
(B)
Next instrument is the TissueTek® paraffin
dispenser or Embedder, which is a great
help in embedding the tissues in paraffin
wax or so. The technologist will embed
the processed tissues for the next step in
tissue processing and staining. Tissue is
placed in the cassettes and then into the
baskets for paraffin dispensing. The
following figure (Fig.1-2) is an example of
Fig. 1-2: (A) the different types of tissue
cassettes used in tissue embedding, and (B)
a Sakura TissueTek® wax dispensing
system: courtesy of Rankin Biomedical
Incorporation®.
In this department (pathology) the next
instrument candidate to mention will be
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microtomes. Ultra-microtome uses in tissue
processing, cutting and sectioning of tissue
blocks in Scanning and Transmission
Electron
Micrograph/Microscope
(SEM & TEM) and is a research tool this
will not be any further cite here in this part
of the textbook. Nonetheless different types
of normal tissue blocks with different
classes of microtomes for routine microtome
cutting of tissue block, all will be covered in
this part of instrumentation.
The
microtome divides into the following
classes according to the ultrastructure and
function of this instrument and is used for
the purpose of tissue blocks embedded in the
paraffin section and the paraffin section
cutting will be done by these microtomes:
1. Rocking Microtome (Cambridge
Rocking Microtome® is named
for its simple lever action and is
good for paraffin wax embedded and
used in first cryostats. Block size
and consistency matters in this type
microtome)
2. Rotary Microtome (most popular,
and is good for routine works.
Flexible with tissue size and tissue
hardness/tough consistency it is
known as Minot microtome after the
inventor).
3. Based sledge microtome.
4. Ultra-microtome (for SEM & TEM)
5. Sliding microtome and,
6. Rotary rocking microtome.
I will explain shortly the most popular and
flexible microtomes for routine works: the
rotary microtome. This microtome is
capable of coping with hard consistency
tissue blocks and hard tissues and
flexible with their sizes. It cuts at 3 mm with
accuracy and precision. The electric type of
this microtome is used to control the
section-ribbon cutting. The blade of
microtome that cuts the tissue paraffin
sections is disposable ones and need no
longer to do stropping and or strapping (and
honing) for routine cutting with heavy duty
blades, just like cryostats it has antiroll
plate that prevent rolling of tissue ribbon
section back on the knife or this is especially
true with frozen sections on cryostat. The
new blades and microtomes have magnetic
blade that attach with the microtomes
knife holder or chuck and cut accurately
and precisely, the tissue sample in paraffin
blocks will be held in the paraffin block
chuck. Additionally, the blades are sharp to
the extent that they can easily cut section to
thickness of 2-4 μm from a tissue block.
Figure 1-3 shows a new class of rotary
microtome.
Fig.1-3; shows a rotary microtome during
operation, courtesy of Leica biosystem®.
Microtomes are used for cutting tissue
sections for determination and diagnosis of
the diseases by histologic studies.
Caution! When operating any microtomes
caution must be exercised not to trap the
operator’s finger(s) between the blade and
the chuck this mishap can be disastrous and
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may result into severing of the hand and
digitals of the operator.
The next instrument is the cryostat that has
an important function in the surgical
pathology lab and need a thorough
understanding of its operation (Fig. 1-4,
shows a cryostat microtome).
Fig. 1-4: shows a cryostat microtome for
frozen section and emergency/stat tissue
section such as malignant tissue submission:
Courtesy of Leica Inc®.
This is used for frozen section where the
emergency or urgent tissues are cut and
prepared in sections and stained rapidly so
that the result will be ready for critical cases
in operating room (OR) or emergency
room (ER). The cryostat tissue sections
tissue while it’s frozen: it will be cut thinly
to the aforementioned sizes for rapid,
urgent diagnosis (on-table diagnoses):
the tissues depending on their type require
following temperatures
by different
methods:
1. Liquefied nitrogen (-190°C)
2. Isopentane cooled by liquid nitrogen
(-150°C)
3. Carbon dioxide (Cardice) at (70°C)
4. Carbon dioxide gas (-70°C) and,
5. Aerosol spray (-50°C)
The last pieces of equipment in the histology
and histopathology lab (and cytology) is
the new version of cytology cell sorters and
processor such as T-2000- & T-5000® thinprep and thick prep processor for
gynocologic
and
non-gynocologic
cytology
slide
preparations,
(for
Papanicolaou/pap smears and other
body fluids). Relevantly these cytology cell
sorters usually are manufactured in such a
way that are capable of distinguishing the
prepared slides of different cytology and
histology nature and normal cells,
although cannot distinguish and identify the
abnormalities.
This
cytopathology
advancement has been cited here just for
presentation and is not the concern of the
histopathology works, cytology and
cytotechnology
is
a
separate
specialty/discipline
in
laboratory
medicine program (lab techs) as with
clinical genetics, this current textbook is
focused in general practice specialty in
laboratory
medicine
program
(generalist specialty). Although laboratory
technician are trained for some aspects of
cytotechnology and cytology, but, I escape
this heading.
Safety in histotechnology and
histopathology lab (Kennedy A et al
& Paget et al)
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Safety is always a refresher topic in clinical
and anatomical pathology, which need
special attention while working in this
section of laboratory. This is due to
sensitivity of the job especially when we are
dealing with cuts, lacerations, infections and
or mutilation of parts or burn and
explosion. When we are in the pathology
laboratory all these mishaps may happen and
need a full attention during work and
practice. The PPE (personnel protective
equipment) and other protective
alternatives must be in place to reduce the
chances of becoming ill or injured.
In risk management anything which is
unlabeled or unidentified such as jars,
vials or bottles, solution containers should
be separated and later disposed. Chemical
inventory has its own requirement in
which the chemical should be categorized
according to the chemical reactivity,
corrosives, explosives, acids, and bases
must be stored in the chemical cabinets
according to the height and weight of the
vessels. The heaver solutions containers
should be stored at the back row of the
cabinet and the lighter ones at the front row
in lower shelves. The management and
classification of the chemicals should be
applied for chemicals that need to be stored
in such a way that reactive chemical should
not be in storage with flammables and
ignitable chemicals, acids must not be
stored with the bases and the other
halogens. The glassware such as beakers
and cylinders should be stores in the lower
levels of the cabinet. And taller vessels must
be placed at the back row and the smaller
containers (e.g. as beakers, vials and flasks,
pipettes) stores at the front row, it is
advisable that when opening the dried
picric acid jars, due to the explosive
nature of this substance and stain at that
stage (dried form beneath the lid). There
might be explosion because of improper
handling of the dried jar lid, exercise caution
in handling of this chemical and mercuric
chloride
[including
azide
solution
(containing nitrogen atoms): do not drain,
or pour down the sink. There is possibility
of exploding in the sink drainage tubing] in
preparation of some stains formula can be
dangerous due to their explosive nature
and other incompatible chemicals. The
chemical handling must be done in a fumehood in an isolated area of the department.
The same precaution must be in place for
other acids, alkalis and other class A, B, C,
D (division 1 & 2) E and F hazardous
chemical and biological classifications
(WHIMS). The same should be done and
consulted with manufacturers’ MSDS
(Material Safety Data Sheets) and
WHIMS [Work Place Hazardous
Information System (refer to Part 1,
clinical chemistry and hematology part 2 of
this textbook)]. The laboratory must have
chemical hygiene plan and schedules to
follow.
Of the most toxic, corrosive and
carcinogenic and teratogens used in
pathology department some of a long list
may be named as Dioxane: a dehydrating
agent, hydrochloric and sulfuric acids used
in dye’s formulary, and benzene and
carbon tetrachloride (CCL4) as toxic and
narcotic
dehydrating
and
clearing
respectively.
Some
advantages
and
disadvantages of several chemical used in
pathology faculty will be further
discussed under tissue processing section of
chapter two part five/current part.
Microtomes blades and knives should be
placed with the edge down in the blade
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holder, during waxing of the microtome’s
knife care must be exercised not to chip the
blade and injure you. In case of stropping
and honing (strapping) the heavy duty blade
caution must observed to prevent incising
hand or inflict other injuries to the person
performing the stropping
although
disposable blades are more popular now. As
aforesaid, the microtome operation must be
taken serious for possibility of trapping hand
and fingers between the blade and tissue
holder chucks (vide supra).
Glassware although mostly are disposable
and of the plastic ware types, and these are
replaced for glassware nonetheless if in any
circumstance glassware (with tendency to
break) used it will be a source of tissue
injuries to the operator. It is recommended
to use plastic ware in all time if feasible.
(Turgeon M L et al).
During performance-lines of fixation,
dehydration, clearing and staining (tissue
processing) always it is a good practice to
wear appropriate PPEs including goggles,
masks to reduce exposure to toxic gases and
fumes of the fixing and dehydrating and
clearing agents such as formalin, alcohol
and acetone, etc. Exposure to these toxic
gases can be hazardous in the pathology
setting and should be maximally harnessed
by limiting exposure by utilizing a safer
chemical. Some of the dangers lure in the
pathology labs are irritants, corrosives,
sensitizers,
carcinogens,
teratogens,
toxicants, highly toxics, oxidizers and
biohazards.
During body and corporeal and arterial
embalming at autopsy department care
must be used to prevent cut or injuries to the
technician/technologist or the pathologist for
there are accident reports of infection and
mortality due to careless and sloppy autopsy
performance.
Solution preparation for stain must be
considered and radiation control by
intuitions/intelligence must be in place to
prevent mishaps and risky situations: it’s
ideal to have a thought for a second time
before performance of a risky procedure. It
is also recommended when severing the
bones for bone pathology to consider when
big chunks of bone either biopsied or
autopsied, be severed with appropriate tools
and caution be taken due to toughness nature
of the bone before fixing the desired
sections and cutting.
radiation emission sign and
biohazard signs must be posted in all
The
blood, body tissues and body fluids areas
(where
biohazard
&
radioisotopes
manipulation are engaged) and in radiation
emitting areas, when staying in this area
the personnel must wear radiation badges or
dossier. The following signs are the
radiation and biohazard signs for restricted
areas (Fig. 1-5), isotopes are used in
conjugation process to trace metabolic
events in tissues. They can be conjugated
with enzyme, antigens and antibodies to
generate signal and detect proteins and other
metabolites of interests in tissue sections.
The
clinical
pathology
and
anatomical/surgical pathology labs must be
restricted only to authorized personnel.
The area must be periodically survey with a
survey-meter/dossier or radiation detectors
(GM tube/Gieger mulller tube/counter) and
in cases of critical radiation threshold,
radiation containment team must be
summoned.
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(A)
(B)
Fig. 1-5: (A) biohazard signs indicating
presence
of
possible
biological
contamination, and (B) the radiation
emission hazard, indicating danger of
exposure to fast penetrating radiation energy
such as radioisotopes.
In these departments food, beverages and
smoking is prohibited, hair should be tied
at the back, chewing on pens are not allowed
and hands must be off the face to prevent
accidental contamination either from the live
or fixed tissues, and the books and
belonging related to the lab must be
minimized. Care must be taken when
severing or grinding, mincing and
centrifuging to prevent aerosolization of
materials from the tissue /fluid particles.
HEPA (High Efficiency Particulate Air)
filter in biological safety cabinet must be on
and the sash half open during tissue
manipulation: working with these materials
must be performed under the safety
hoods/safety cabinet and the section premise
must be assisted with a proper ventilation
system called HVAC (or Heat Ventilation
with Air Conditioning) aside from hoods
and safety cabinets. This is to clear and
circulate the internal atmosphere and
aeration of the lab space, the section must
have negative air pressure to prevent escape
of infectious particles from the entrances of
the pathology section to outside, by the
availability of the negative pressure inside as
the air current flow inside once the entrances
are open. Thus this prevents escapes of the
air through the doors and air conditioning
will be handled by internal ventilation inside
the lab section just to prevent dissemination
of infectious particles.
Engineering controls of premise and
ergonomics are important aspects to
personnel’s states of health: these should be
used maximally to help maintain the hazards
associated with lack of these principles. To
counter-intelligent the problem we should be
prepared for any mishaps or accidents
occurring in the lab space and act by training
in the first-aid courses/programs. The
spillage of the toxic and chemically and
biologically hazardous substances
must be contained by means of chemical
and alkalis neutralizer kits and
biological spill kits available on site
respectively.
Chemical disposal of
hazardous substances must be handled
efficiently and by a trained personnel and a
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waste
disposal
management systems. Before disposal all
contract
with
biological wastes and chemical wastes must
be collected and disinfected on site through
autoclaving and storing in an isolated
segment of the lab along with container of
chemical wastes and finally by removal by
the external waste management
systems contractors.
Some substances can be washed and drained
in the sink: i.e. detoxified formalin in prior,
or acid and bases can be neutralized and
subsequently disposed or drained in the sink.
However all chemical must be stored and
removed according to the OSHA/OHSA
(occupational safety and health
act/agency) and local regulatory bodies.
Description of all chemicals safety issues is
out of the scope of this text and for more
information refer to the sole textbook
references at the end of the part five (current
part) and MSDS with WHMIS. Personnel
must be acquainted with the location of
safety blankets, showers and eye-fountains
for the emergency burn, fire or eyes
affliction due to chemical spillage and
splashes.
Working with cytospin and tissuecentrifuges or other type centrifuges must be
done with lid closed and essentially not to
use the break (this to slowing down its
velocity). The nails and hairs must be short
or as said tied at the back. The safety plan
must be reviewed periodically and personnel
awareness of the regulation must be trained
at certain periods and these programs have
to be documented to update the program
schedules and the training contents.
Incident report is essential for the
prevention of the same occurring problem
and has to be presented in a format or form
that contains comprehensible information of
the accident be reflected in that report.
Report keeping and document maintenance
is indispensable for further referral and
compliance with government agencies and
US department of health and human
services and Health Canada. The
following is the figure (Fig. 1-6) of cytospin
assembly for smear preparation.
(A)
(B)
Fig. 1-6: (A) schematic presentation of a
cytospin, courtesy of springer-images®, and
(B) the actual centrifugal bowl of cytospin
570
detached from the main centrifuge set and
note the slides position in for absorbing
materials from the centrifugation, courtesy
of thermo-scientific®.
Collection and reception of
specimen and preparing for
specimen process in front end
aspect (Bancroft et al)
The first point where the reception starts in
pathology lab works is the receiving of the
specimen after arrival at reception: the
specimen should be either receives in jar,
vials with proper amount of fixatives or it
may be in the form of fine needle biopsy or
touch print on the slide/smear with alcohol
or other preservative/fixation aerosol
sprayed on it so that it is fixed.
The identifiers of the specimen must be
checked as name of the patient, birthdate,
address and the third: hospital or institution
number including the ward it is originated
(OR, ER and CCU or the mortuary etc.) with
MRN (Medical Record Number) attached
and written on the pathology Requisition
Paper and on the bottle or jar or vial and
other vessels where the specimen is fixed (as
label) with sufficient amount of fixatives
and have screw-capped. The labels may be
computer generated with bar codes or
manually written, the labels must be placed
onto the requisition paper and the vials for
further processing. The amount of fixatives
must be 15-20 times of that specimen, and
the container must be screw capped as
mentioned.
The sample collection and reception from
operating room as surgical specimen will be
in form of whole or part of an organ, or
another specimen from regular minor
surgeries in O.R (operating rooms)
including biopsies. Or the specimen may be
from nursing units as in pleural fluids or
other fluids (rarely) and or originates from
out-patient department (OPD): lastly these
specimens may be referred from postmortem department (morgue).
There are few formats that specimen arrives
in the histology lab whether it is fixed
with 10% solution of formalin in a clean
non-breakable vessel, 2nd in form of wax
block third as in unstained section on smear
and lastly stained sections on slide.
All
specimen
arrives
in
the
pathology/histology department must
be fixed with 10% formalin excepts for
bacteriological,
virological
and
toxicological specimen that should not be
fixed, other specimens’ fixation must be as
quick as possible after death or biopsy to
prevent tissues putrefaction and decay and
the specimen in the jar must have proper
labeling which its info must match with the
requisition.
Forensic specimen for chain of custody
must be sealed in every step to prevent
tampering with it. And lastly for electronmicroscopy the specimen should be fixed
rapidly
with
glutaraldehyde with
phosphate buffer at PH 7.2 to 7.4.
After reception and fixation and checking
all criteria for proper reception, it must be
given an accession number, name, date of
fixation and type of tissue, physician name,
diagnosis, patient’s location whether in the
ward or OR (Operating Room) or ER
(Emergency Room) must be indicated on the
label and request paper along with request
for the type of processing such as frozen
section or routine processing.
571
Subsequent to all these it should be given
accession number and the data should be
entered into the accession log manully or
electronically. All data must be entered in to
the manual logs or computer terminal.
Specimens that do not conform with the info
(identifiers) written on the vessel’s label and
request paper must be checked before
discarding if the specimen is of critical and
urgent nature/value therefore it must be
consulted with the ward and persons in
charge and come to the advice of supervisor.
The non-urgent specimen should not be
accepted at all due to discrepancy and must
be discarded. Specimens that arrive at the
weekend where regular personnel (day
shifts) are not available must be left in a
moistened cloth or gauze with normal saline/
NSS (normal saline solution) and left in
fridge temperature until the regular shift
starts, if the specimens are small enough.
The specimen should never be frozen or left
long at surrounding temperature or hangup to dry and shrink. It must be optimally
and immediately be fixed, preserved and
ready for processing.
electrolytes and its pathology in clinical
chemistry part one of the textbook) and thus
results into hardening of these tissues. In
connection to preparing for specimen and
processing, stains and dyes must be filtered
before staining is performed. Alcohol
solutions/dips for dehydration must be
changed on periodic schedule and acetone or
other clearing agent must be changed and
refreshed on certain timescale. Buckets for
washing and rinsing of the stains and the
slides must be changed as well at the end of
every batch or run. If using tissue processors
for processing maintenance is important this
is consistent with washes/flushes with
xylene followed by an alcohol rinse: this is
usually
done
automatically.
Toxic
hydrocarbons generated in the air and tubing
by the tissue processing in the tissue
processor must be cleaned by carbonfilter(s) assembled/build-in to counter act
the accumulation of these noxious
substances and their vapors.
Bone specimens must be severed and sawed
and then decalcified after fixation. Calcium
ions must be removed from tissues
containing this ion. Example of these
calcified tissue are atheroma tissues, bone
(calcified), teeth and tissues that sustain
damaged (necrotized) and are classified as
epithelium and thyroid tissues, cardiac
valves and kidneys, etc. These tissues
harden and calcify due to resorption and
deposition of calcium ions and other salts:
calcium ions along with phosphates in
tissues such as dystrophic calcification,
metastatic calcification and calcinosis. This
process is a quite complicated, however in
short is due to imbalance of calcium ion in
the tissues (refer to calcium ion as
Fixation and fixatives used in
Histotechnology (Bancroft et al)
Chapter Two (2)
F
ixation comes before any other
processing of tissues and in bone
decalcification which all fixations
come first. The purposes of fixation
are to prevent the tissue from autolysis,
putrefaction and decay for a better
observation under the microscope,
the
preservation and hardening the tissue,
solidifying the colloidal materials and a
better visual or optical differentiation of
structures. There are many ways of fixing a
tissue section as examples we have heat,
chemicals and microwaves treatment or for
572
some tissue fluids and secretions by means
of alcohol and air.
The most important substances that can be
fixed and stained to screen for their tissue
pathology are proteins, lipids (depending on
like frozen sections), and carbohydrates that
may be found in different combination in
different tissues. These chemical substances
come into combinational interplay so to
create molecules of different shape, size and
properties
such
as
sphingomyelin,
sphingolipids, lipoproteins or phospholipids,
etc, fixing of these substances are crucial in
histopathology:
the
most
important
substances in histopathology, however, are
proteins from the histopathological
aspect/perspective or point of view. Proteins
are the main building blocks of all
substances such as proteins, enzymes,
glycoproteins,
lipoproteins
and
proteoglycans, etc.
Fixations have profound effect on the next
stages of tissue processing as it may affect
dehydration, clearing and staining.
Fixation can affect lipid and this is almost
always lost during tissue fixation, on-thecontrary, there are some treatments before
fixation that can take place to preserve and
save lipids somehow.
Fixatives are divided into aldehydes
(formaldehyde,
glutaraldehyde,
etc.),
oxidizing agents (osmium tetroxide,
potassium permanganate and potassium
dichromate) and protein’s denaturing
agents as alcohols and acetic acid. Also
mercuric chloride, picric acid and heat and
microwave are other substances and
methods to fixing tissues.
In microwave technology fixation of tissues
can cause oscillation of biopolar
molecules in interaction with microwaves.
This idea will be extended under microwave
fixation later. However, the interaction of
chemical fixation with DNA and RNA
requires heating to 65°C and 45°C
respectively to uncoil these molecules for a
better fixation and staining. Nevertheless it
does not react under normal thermal
condition (room temperature, etc.). Similar
situation is observed with glutaraldehyde
as well. In Carnoy’s solution, alcohols
such as methanol and ethanol are used as
fixatives. These alcohols can uncoil DNA
and therefore preserve it.
As said lipids usually are lost during fixation
even by the use of glutaraldehyde, this
fixative can fix phospholipids with binding
to some definitive amino groups.
Nonetheless formalin has been shown to fix
unsaturated fatty acids: lipids may be
examined with frozen sections.
Carbohydrates fixation may accomplish
as well with the use of alcoholic solutions
and
are
recommended.
Alcohol
formaldehyde is better fixative for human
skin tissue than neutral buffered
formalin (NBF). At last, the use of
fixatives-cocktail or mixture has some
advantages and disadvantages in fixation of
variety of tissue sections, such as
glutaraldehyde-paraformaldehyde
for
electron microscopy or glutaraldehydeosmium tetroxide for the same purpose.
These cocktails may produce artifacts in
staining process and it is better to be used
sequentially especially glutaraldehydeossium tetroxide.
Fixatives may coagulate tissue (and
proteins) to some extend however it may not
affect the examination and tissue processing:
it may have a none-coagulating property
573
or a coagulating property. The nonecoagulating fixatives cause a little distortion
or breakage of the tissue called “soft
fixation”, however, the coagulating one is
capable of hardening the tissue and is
breakable or fragile it is known as “hard
fixation”.
popular fixatives is 10%
formalin which is cheap and easy to
prepare, and is the best fixative for the
nervous system, it does not cause so much
hardening of the tissues. Also fixation with
10% formalin staining can be easily
carried out, tissue block preserved by
formalin does not require washing before
processing along with others properties as it
penetrates the tissue reasonably well and
natural color of the tissue can be restored.
There are a few disadvantages for 10%
formalin
(40%
formaldehyde
and
NBF/Neutral Buffered Formalin) as it may
cause dermatitis and asthma and as an
irritant to the skin and nostrils. Old unbuffered formalin may cause dark brown
pigment(s) due to buffered formalin in
bloody tissues (artefacts). In addition the
hematin
with
combination
with
formalin/paraformaldehyde may form dark
pigments of Acid Formaldehyde Hematin
(AFH) nature: spleen and liver and blood
vessels (blood forming tissues) are the most
likely candidates for this problem, it may be
removed by saturated picric acid
solution. This is an undesirable situation
for tissue sections and can be troubleshot
and prevented by buffering the fixative
(formaldehyde).
The
most
The chemical fixation as formaldehyde may
have certain effects on staining that is these
combination may act as mordant: this is
when the tissue will look more brilliant due
to action of dye and tissue in selective
staining.
In reception of the tissue and unlikeliness of
fixation and if the tissue section is not fixed
immediately cover the surface of the tissue
precisely with gauze and moisten it with
NSS and leave it in the fridge until attended.
Fixative volume must be 10-20 times the
size of the tissue/specimen. In this regard
fixatives are toxic and once come in contact
with skin or inhalation or swallowing it may
irritate and damage tissues once this occur
wash the skin with copious amount of water,
and seek medical attention for the other
routes as per se . Their vapor might be the
source of toxicity and a well-ventilated
room is essential as well. In this connection
we can use biohazard cabinet and fumehood for biohazards and chemical hazard
purposes respectively.
Other fixatives such as mercuric chloride
may bind to some amino acids residues in
proteins to fix the tissues and it is used as a
secondary fixative (post-fixation). Postfixation of mercuric chloride of ultrastructural preservation would be poor
nevertheless use of trichrome staining
works well in this regards many immuneperoxidase methods are satisfactory.
As with Microwave fixation, it may
produce poor staining due to under-heating
the tissue or overheating (>65°C) may cause
tissue
distortion
and
vacuolation.
Therefore an optimum temperature is ideal:
this may be from 44-55°C. Poor quality
sectioning is a cause of under-fixation with
under-heating. In over-staining due to
overheating the cell cytoplasm will be overstained and nuclei may be pyknotic. This
utility of microwave in fixation process is a
good tool in routine fixation and processing
574
with well tissue preservation and thus is
adequate.
The use of routine fixation with
microwave give reasonably good result in
many staining procedures or methods such
as neuropathology with Bielschowsky,
Bodian
and
Nissl
methods
or
immunohistochemistry applications and
enzyme histochemistry including several
others. Microwave technology can be used
in electron microscopy with osmium
tetroxide fixative or with some other
cocktail fixatives (vide supra). In this
method post-fixation with microwave may
produce a satisfactory result.
In respects to the factors that may affect the
fixation of tissue, we have hydrogen ion
concentration, time and duration of fixation,
penetration of the fixative into the tissue,
osmolality of the surrounding and
concentration of fixative and the
temperature, all can be critical: in short,
duration of fixation is critical when too
lengthy fixation may result into overstaining and distortion while under fixation
is the result of under-staining. The
fixation’s effects of PH are the best shown
in optimal tissue fixation between PH= 6-8.
Outside range is detrimental to the tissue
processing. Some procedures as detection of
adrenaline and noreadrenaline mandates
specific PH as PH=6.5 for chromaffin
reaction. Other example for specific PH is
the gastric mucosa preparation and optimal
fixation that needs to be fixed at PH= 5.5.
As for penetration factor, the tissue size is
important: the thinness or the thickness
must be considered for fixative penetration.
It also matters that tissue packing and
cellularity affects penetrations of the
fixatives (as kidneys and pancreas),
dehydrating agents, clearing and
impregnating wax and even the stain.
Example of the tissue size: large organs as
uterus must be toast rack and spleen must be
opened and sliced thinly. For brain tissue
with
thick
grey
matter:
the
neuroanatomical relationship must be
maintained by preserving whole organ
suspended in 15% NBF (normal buffered
formalin) for 3 weeks to obtain a
satisfactory fixative-tissue penetration
In addition for osmolality the hypertonic
or hypotonic solutions may rise to tissue
shrinkage or swelling accordingly (hypoand hypertrophy respectively). Isotonic
properties are likely optimal.
Fixative concentration of 10% for
formalin and 3% for glutaraldehyde may be
ideal but it varies with PH of the vehicle.
The last factor that affects the tissue fixation
is the temperature during which the tissue is
fixed. Room temperature is carried out
for routine fixation for surgical pathology
specimen however electron-microscopy
(EM) may be fixed between 0-4°C with
exception of some tissues under this kind of
study: i.e. mast cells need room temperature.
Also frozen sections mandates for
emergency situation applies with freezing
temperature (-20°C or lower). Other factor
may be in need during tissue fixation and
processing such as the use of detergents,
substances added to the vehicle and changes
in volume of the fixative solutions.
During fixation of tissues we may use a
primary and secondary (post-fixation)
fixation with some fixatives may produce
artifact as well such as pigments or
precipitates with the formalin’s artefacts
(vide supra): these precipitate have already
been discussed.
575
Of some others artefacts, delocalization
of artifact or the precipitates to other parts of
tissue sections (spreading) is also likely, a
good example for delocalization (false
localization) is the effect of glycogen and
enzymes to other part of the tissue section:
(vide infra/enzyme section) these are called
“streaming artefacts.”
Fixatives
as mentioned vary and are for
different purposes, fixatives such as
formaldehyde fixatives are good for
routine purposes, and it can preserve almost
most of the molecular species in the tissue
sample
with
different
intensities/concentrations. Carbohydrates,
proteins and lipid may be fixed properly.
The different formulas are used in formalin
preparation to fix tissues. Examples are
formal saline, alcohol formaldehyde, formal
calcium, and NBF (neutral buffered
formalin). Other kinds of formula exist for
tissue fixation such as paraformaldehyde
and phenol formaldehyde. Out of alcoholic
formaldehyde, the Carnoy’s fixatives are
the best for preservation of glycogen. Once
the alcohol has been added to formaldehyde
it increases the fixability character of the
fixative solution for a better fixing. As the
names of different mentioned formaldehyde
solution indicate these are added with
particular substances such as saline, alcohol,
buffer, calcium or phenol etc., these
augment the fixing abilities of solution and
make them more specific in action (Bancroft
et al).
Picric acid solution have different formulary
such as Bouin’s with glacial acetic acid
added, or Gendre’s fluid with different
percentage of formaldehyde and picric
acid solution plus glacial acetic acid, also
the last but not the least is Rossman’s
fluid (most popular for carbohydrates) with
picric acid saturated with alcohol and
neutralized formalin. These are good by
binding to histones and proteins and
glycogen as well. The addition of picric acid
solution to the formula adds and conveys a
yellow color hue to the tissue section and
may be removed by treatment of lithium
carbonate or another acid dyes.
Mercuric chloride fixation formularies
consist of a few fluids such as Helly’s
fluid with potassium dichromate and
mercuric chloride, or Susa’s fluid added
with trichlroacetic acid, dichromate and
glacial acetic acid. The buffered
formaldehyde sublimated contains
mercuric acid sodium acetate and
formaldehyde and lastly among the mercuric
chloride formulas are Zenker’s fluid, with
some potassium dichromate, formaldehyde
and glacial acetic acid contents. Mercuric
chloride fixing fluids utilizes in fetal brain
anatomopathologic examination. It has
poor penetrability and produce black
pigmentations (except Susa’s) are its
disadvantages: pigments can be removed by
0.05% iodine solution before staining
(during deparaffinization).
As in protein demonstration NBF and
formaldehyde vapor can be used and for
lipids, Baker’s formal calcium is used for
phospholipids and for general lipid
screening cryostat section (frozen sections)
are adequate although in routine and in most
cases lipids are lost during processing.
Elfman’s fluid is good preservative for
lipids in electron microscopic examination.
It contains mercuric chloride and potassium
dichromate.
576
For general purpose carbohydrates fixation
and demonstration, Rossman’s fluid and or
cold absolute alcohol are suitable.
Immunohistochemistry
and
immunoenzymatic methods has the same
principles that have been discussed in the
clinical chemistry section part one, although
the
principles
may
apply
to
histopathology and histotechnology a
little differently. Tissues are processed and
enzyme or antigen/antibody tagged tissues
will be studied under microscopes by
fluorescence, or labeled or tagged
enzymes-tissue with radioactive substance to
trace required metabolic activity or
substances or tagged with immunologic
signal
chemicals/molecules
generating
(e.g.
immunoperoxidase,
or
other
antigen/antibody conjugates labeled with
enzymes or labeled isotopes) may be used to
detect proteins, carbohydrates and lipids,
such as antithyroid antibodies or
antinuclear antibodies.
Concerning immunohistochemistry fixation,
fixation is used for few seconds with
formaldehyde and tissue may be prepared by
paraffin sections. As with enzyme
histochemistry fixation in any form is
not recommended: only each enzyme’s
detection indicates its own characteristics:
this should be considered. Therefore for
general purposes we can use 4%
formaldehyde or formal saline overnight
and do frozen sections. Generally frozen
sections and cryostats are ideal to study
enzymes some may need to be fixed in
acetone or formaldehyde. Scanning
Electron Micrography (SEM) and
Transmission Electron Micrography (TEM)
are used for research purposes and is not
cover in here part five, which advocated for
routine light microscopy. Flowcytometry
is used in histopathology department for
determination and identification of peculiar
substances.
Organs as brain, renal biopsies, GIT
(gastrointestinal tract), liver, eyes, lungs,
heart and lymphoid tissues/ and nodules or
laparoscopic specimen and other organs
must be fixed and slide sections prepared for
further study in paraffin blocks. For example
eyes and liver may be fixed in buffered
formaldehyde or kidney tissue sections
(percutaneous) may be preserved in NBF for
routine embedding. In regard to EM studies
(electron-microscope) tissues may be fixed
with
glutaraldehyde.
Buffered
formaldehyde and NBF are routinely used
for different organs fixation.
Application of microwave and
tissue processing (Leong et al, Shi et
al & Bancroft et al)
In 1991 Landmark discovered that antigens
can be retrieve by utility of microwave,
special stains can be introduced after tissues
safely were fixed and sustain with no
damaged subsequent to microwave
treatment. This application practically do
not break or damage bonds between
molecules and safely delivers fixation to the
tissue in which optimal staining can be
obtained. The main operating scheme has a
built-in magnetron which produces the
microwaves inside the microwave oven. The
waves are in pulsate manner with hot and
colds characters. This disadvantage is
remedied by carousel or rotor built onboard to counteract the pulsate waves of the
oven to expose the specimen to constant
heats. This method distributes heat and thus
the wave equally to every section of the
tissue and therefore fixation may take place.
577
Principles involved are the dipolar or any
polar molecules in protein or other chemical
with the same property once hit with these
waves they (these molecules) can rotate to
180° with a force of 2.45 billion cycles, thus
heating up the specimen. This heating up
helps to fix the tissue with different
components easily and fast.
The advantages and uses of microwave
technology in histotechnology include 1)
low volume of specimen needed while in
conventional fixation tissue volume is
much more than the microwave technology,
2) short time of processing and fixation, 3)
used in Masson Fontana method for silver
staining of tissues with rapid fixation and
processing these also facilitate silver
impregnation, 4) demonstrates other
components with other methods such as
Periodic Acid Schiff (PAS) and Perl’s
Prussian Blue (PPB), and 5) can be used
for enzyme chemistry such as ACP and ALP
(acid and alkaline phosphatase) and others.
Some of the disadvantages are: the
temperature should be adjusted so that to
accommodate faster result. For getting a
better result either the time must be lengthen
or voltage/power be increased. In this
volume of solution must remain constant.
The maintenance includes periodic checks
for the instrument, and some of the solutions
are not usable after usage, e.g. Perl’s PB.
There must be stirring the mixture to
produce convection current, this is due to
pulsate heating of the contents (vide supra).
There is probability of precipitation of sliver
in cases of too long standing in microwave
of silver. At last, there might be some
explosion if treated with some chemicals
such as chromates and chromic acid.
The applicability of the microwaves has
been demonstrated by heating the striated
muscle (roasted beef) sized and cut 2x2x2
cm 3 in 100% ethyl alcohol at 70°C: one
section placed in microwave and other
placed in common and conventional
ovens over 5 minutes fixation. The beef in
the microwave showed gray and all the way
dry through its thickness while the one with
ordinary oven show only surface grayness
and the core was red and unfixed (J. D.
Bancroft et al). The following picture (Fig.
2-1) shows microwaves treated fixation in a
human tissue.
Fig. 2-1: On the left (pictures: A, C and E)
are formol fixed tissues, and on the right
(pictures: B, D and F) are fixed with
microwaves, note the clarity of tissues
sections as corneous layers in subsequent
pictures.
In addition to the above advantages core
biopsies/biopsies may be done with less
than 30 minutes of operation of the
microwave and tissue section smaller than 2
mm in cross section may be done less than
one hour. This is much faster and better than
any fixation in tissue processors. Antigen
578
retrieval can be done quickly by microwave
processing and addition of citrate phosphate
buffer to the tissues. This leads to
unmasking of antigen in the tissues (Leong
et al).
Tissue Processing (Paget GE et al)
Following fixation (this is antecedent to
decalcification of bone tissue/slabs and
the first step in all tissues processing), we
have to dehydrate the tissue block that
have been fixed this may be accomplished
by using chemical agents to take out or
extract the water molecules from the
aqueous solution of fixed tissue. The
dehydrating agents are 1) ethyl
alcohol/ethanol (with concentration gradient
70-100%, which is the most popular and
common tissue dehydrant: it shrinks the
tissues and is miscible with water and
replaces water entirely: this is somehow
inexpensive dehydrating agent, 2) butyl
alcohol, a slow dehydrant and miscible
with paraffin but causes less shrinkage of
tissues, 3) acetone, is a volatile and
flammable dehydrant with strong odor, 4)
methanol, may be a substitute for ethanol, 5)
isopropyl alcohol, a good substitute for
ethanol, may cause shrinkage and hardening
of tissues section, celloidin is insoluble in
isopropyl alcohol and cannot be used for this
purpose. There are other dehydrating agent
conventionally used in the past and are not
used in histolab any longer these are 1)
ethylene
glycol-mono-ethyl-ether
(Cellosolve); it is hygroscopic and no need
of graded solution, 2) tetrahydrofuran;
quickly evaporates and is a nontoxic agent,
3) dioxane (no longer in use in
histopathology labs), it is toxic and causes
tissue distortion.
The factors involve in proper dehydration
are: 1) the nature of dehydrant, 2) the
fixative in use, 3) the number of the pieces
of tissue suspended in the cassette, 4) the
size and type of tissue (density), and 5) the
volume and the amount of dehydrating
solution. The inadequate fixation and
dehydration ultimately results in insufficient
clearing and wax impregnation. Therefore,
it is a must to optimize all tissue
processing from the time of fixation to
dehydration,
clearing
and
waximpregnation. To have a well stained
tissue section the followings may be
involved and controlled: the tissue section
may have incomplete dehydration due to
tissue thinness or thickness, the inadequate
immersion into the dehydrating agent as
timing schedule(s), or low volume
dehydrating agent, next would be the size of
tissue as too many pieces of tissue blocks
may hinder the proper clearing and staining,
also the erring in alcohol gradients
(grades) may affect the clearing stages, and
lastly the expiration of the strength of
alcohol or dehydrating agents may result
into improper staining.
Next step in the tissue processing would be
clearing there are several clearing agents
used for this purpose, the purpose of
clearing is to increase the brilliancy, clarity
and augment the optical density and render
tissue transparent. If the clearing is
insufficient, it would ultimately affect the
wax-impregnation and embedding or
staining quality. The agents used for
clearing include namely as: 1) xylene, this is
the best and it is rapid and easily clears from
the wax, it’s somehow a bit expensive,
however it is flammable and mild irritant, 2)
benzene, it is a rapid clearing agent with
minimal tissue shrinkage however, it is
carcinogenic (very toxic), flammable and
579
rapidly volatile, 3) toluene, is a rapid
clearing agent, with quick elimination from
wax, only alcohol must be used as
dehydrating agent although the fumes are
toxic and it is narcotic in nature, 4)
chloroform as a clearing agent is good for
large size tissue blocks, nonetheless it is
toxic and is anesthetic naturally, it is
hydroscopic as absorbs the air moistures, 5)
methyl benzoate, it is a tolerant clearing
agent and clear quickly however it is toxic,
6) cedar-wood oil, extremely tolerant
clearing agent and does not extract aniline
dye, but it is a slow agent and difficult to
remove from paraffin-wax, and lastly; 7)
carbon tetrachloride or known as (CCL 4
).
As for processing and clearing the tissue
with xylene in spite of unavailability of
xylene the other clearing agents mentioned
may be utilized although there are other
clearing agent called ”xylene substitutes”
their example is as citrus extract,
nonetheless these substitute are toxic with
strong odor and have not been commonly
adopted in histology due to their
disadvantages overweigh their advantages
(Kernan et al).
In handling the clearing agents the
histotechnologist must be vigilant of
drawbacks of these substances and they
should use more environmentally and
physically friendly clearing agent to reduce
their toxicities, they should avoid exposure
of these substance to sun light, also handle
these agent with mechanical instrument than
with sole hands, they may use full PPEs at
all time and keep all caps and lids of the
reagent vessel tight and closed. For disposal
of toxic chemicals consult MSDS and
WHMIS, federal government of Canada
and US have regulatory bylaws and
commission for proper disposal of all toxic
substances
including
histopathology
chemical wastes, recycling may be used
as a way of immediate disposal.
Subsequent to dehydration and clearing we
precede
with
infiltration
and
impregnation, in impregnation, some
impregnation mediums impart strength to
the tissue section these are in forms of wax
so that in microtome cutting the tissue may
have torque and proper consistency/texture
to
prevent
tissue
collapse
during
microtomy. The followings are the most
common impregnating medium, 1) the most
popular and common is the paraffin wax
which available and cost effective, the
temperature of the surrounding may dictates
the type of paraffin wax some are
considered soft wax at 45-50°C and others
are hard wax are at 52-60°C. The best wax
for temperate climates are between 54-58°C
can
stand
this
environmental
temperature with good quality section
cutting. 2) Combination of different wax
such as beeswax, rubber, and polymers are
being used in tissue processing which are
hardeners of the wax. The fact is that
addition of polymers waxes to the paraffinwax will give a uniform texture and a
better ribbon sectioning of the tissue section
3) (undecalcified bone section make use of
MMA/GMM impregnating agents/ refer to
bone histopathology section further ahead in
chapter three/vide infra). 4) There are others
infiltrating and impregnating waxes such as
carbowax and nitrocellulose which I will
escaped explanation in here as the text is
considered routine practice and the most
common wax would be paraffin as cited.
The decalcification of bone subsequent to
fixation and processing will be considered as
a part of tissue processing however I deal
580
with this section in chapter three (title bone
processing and staining)(Bancroft et al).
Specific tissue and body site for processing
and staining may include spleen and lymph
nodes (will be discussed in chapter four
under heading of immunohistochemistry of
query lymphomas), bone marrow,
kidney, trans-bronchial, open lung
biopsies and bronchial biopsies and nerve
biopsies (this will be considered in
neuropathology section of chapter four
histopathologic techniques in neurology).
As
with
bone marrow biopsies and
aspirates, a smear is made from the
aspirates and sent to hematology department
for further processing as well the trefine
portion is retained in the histology
department and fixed either by B-5 or
Zinker’s solution (Bouin’s fixatives/vide
supra) and it should be mentioned prior to
processing, it must be decalcified and then
process either by paraffin section or plastic
impregnation. Due to size of the bone and
care must be taken to prevent overdecalcification. Check frequently [refer to
bone section of chapter four (4)]
As in kidney biopsies the purpose of the
biopsies are either for kidney pathology or
kidney transplantation either in form of core
biopsies or percutaneous sampling as
FNAB (fine needle aspiration biopsy).
In either cases fix the kidneys in formalin as
other tissues and do paraffin waxing as for
other tissues, for electron microscopies
glutaraldehyde may be used: refer to the
fixation at chapter two (2), for cryostat
sections the Sudan red stain may be used.
Concerning the open lung and transbronchial biopsies the need for rapid
processing in cases of pneumonia and
cancer warrants sampling and rapid
processing especially in HIV patients. This
is an urgent or priority, stat specimen
must be acted on appropriately. For urgent
tissue frozen sectioning is ideal and
aspirates may be processed according to the
need as in smear for cytology or imprints:
touch imprints and crush smear are made
from specimen obtained by transbronchial endoscopy. This is done by
applying a bit of tissues obtained by this
procedure and crushed in between the slide
and fixed by formalin fixative and proceeds
accordingly to the standard of practice.
Nerve biopsies will be tangibly covered in
chapter four (4).
The last section in tissue processing is
microtomy and tissue mounting onto
slides. The microtomy is done by
microtome: once the paraffin wax have been
blocked out by TissueTek® processor and
ready, we place the paraffin blocks made by
either tissue processor or manual processing
(TissueTek), the paraffin block will be
placed in the microtome chucks and then
by considering all precautions the
technologist attempts to cut tissue ribbon
sections. This is done by vertical rise and
fall of the microtome blade perpendicular
to tissue paraffin block thus a tissue ribbon
may be obtained/formed. Subsequently the
water bath is used to fish-out the floating
tissues that has been selected form the
ribbons and floated on the water bath. For
further expansion and quality of the fished
out tissue (picking up tissue section)
sections from the water bath, a surfactant
(which increase surface tension) such as
30% alcohol or acetone (1:10-1:5 ratio), or
gelatin may be used in the water bath to
581
expand and dissolve the paraffin wax and
for a better adhesion on the mounting
slide can be used. Sometimes at this stage a
warmer plate may do the melting residual
paraffin wax in the tissue that has been
picked up and this does increase the
adhesion to the slide Then once the selected
tissue section are mounted on the slide it
will be once more now reverseally
decerated/hydrated by immersing the
tissue section into xylene and alcohol grades
accordingly (a reverse alcohol grades of
dehydrating). After this hydrating, we
attempt to dehydrate with increasing alcohol
and acetone grades respectively, just like the
beginning of the tissue processing at the
final steps. Lastly it is stained (vide supra)
and a mountant such as resinous
mountants or albumin applies to the section
and cover-slipped and will be kept for
further studying. The final step in tissue
processing is to assign the accession
number, tissue source and the technologist
initials to the silde (s).
Attention: some of the troubleshooting
points in microtomy includes as followings:
1) When ribbon and consecutive sections
are curved;
-The cause may be the block is not
parallel to the chuck and blade (trim with
sharp scalpel until parallel.
- blade is blunt in one area (use
different part of the blade or replace)
-surplus area of wax at one side
(trim away excess wax)
-tissue varying in consistency
(reorient block by turning 90° or treat and
cool block with ice cube, or mount
individual sections)
2) Alternate sections thick and thin;
-wax is too soft for tissue or
conditions (cool block, or use a higher
melting point wax)
-block or blade loose (tighten the
block or blade)
-insufficient
clearing
angle
(increase slightly the angle)
-mechanism of microtome faulty
(check for the faults, pawls may be worn)
3) Section role in tight coils;
-blade blunt (change blade or use
different side)
-too little rake angle (reduce blade
tilt, if clearance angle excessive)
-section thickness too great for wax
(reduce section thickness or slightly
higher the melting point wax, while
cutting breathe into the section).
3) Section will not make ribbons;
-wax too hard for sectioning
(breathe into or re-embed in lower melting
point wax)
-debris on blade edge (clean with
xylene moisten cloth)
-knife angle too steep or too shallow
(adjust the optimal angle)
4) Calcified areas in the sections;
-nick in the blade (use different part
of the blade or replace)
-hard particles in the tissue (if
calcium deposits; decalcify or remove with
sharp point scalpel)
-hard particles in the wax (reembed in fresh filtered wax): for other
important details of section troubleshooting, refer to the terminal references in
this textbook.
Microscopy (Turgeon et al)
The light microscope or photomicrograph
was discovered by Antony Leeuwenhoek in
1677, the structure has unique properties to
582
study cells and tissues sections (for ideas
how to adjust the microscope by means of
Köhler illumination refer to clinical
chemistry: part one of this textbook). The
microscope is composed of eye piece lenses
(oculars), body tube, carousel with
objective lenses built-in, condenser, stage
with clips and vernier scale, aperture iris
diaphragm, course and fine adjustment
knobs, arm, stage adjustment, rheostat, field
diaphragm, condenser focus knob, interpupillary distance adjustment, diopter
adjustment, source of light, switch on and
off and base or stand. There are different
kinds of microscope that uses in clinical and
pathology laboratory: these are light
microscope (bright-field microscope),
phase-contrast microscope, interferencecontrast microscope, polarizing microscope,
dark-field
microscope,
fluorescence
microscope and electron microscopes
(EMs). In these types of microscope the
light microscope has somehow modified to
adjust to the need of the study, except in
EMs, for example in dark-field
microscope, light microscope has
modified with a special sub-stage condenser
to allow visualization in the dark field or
with polarizing microscope the light
microscope has been modified with
polarizing lenses or filters The following
figure (Fig. 2-2) is the ultrastructure of a
bright-field microscope.
Fig. 2-2: the ultrastructure of a histology
bright-field photomicrograph (microscope)
The illumination system of light
microscope has been discussed in clinical
chemistry section of part one and no longer
will mention in here. There are few term
need to be cleared in microscopy and are the
1) magnification of the microscope, 2)
resolving power or resolution, and 3) NA
or numerical aperture.
The magnification of a microscope refers to
the number of times the objective lens or
lenses can be multiply to the ocular(s), this
means that if high power objective is 40x
[the magnification is inscribed on the
metal body of the lens (-es)], once this
number times to ocular which is 10x we will
have a total magnification of 400x; with oil
immersion lens it is a total of 1000x.
For the resolving power or resolution of
microscope this would be the ability of the
microscope lenses to resolve image. This is
the ability to distinguish the closest distance
two objects can be close together and still be
distinguished, mathematical configuration of
this concept is in term of numbers and
583
indices (ratio). This is NA (numerical
aperture) which is the index or the ration of
this phenomenon of resolving power. The
human eye resolution (resolving power) is
0.25 mm, for light microscope is 0.25 μm
and electron microscope (EM) is 0.5 nm.
In empty magnification there is no clarity or
resolution between the two objects close to
each other and they may appear as
dumbbell. The most important of
microscope is the condenser and I will
explain a little about this structure in the
microscope. Condenser (for the microscope
image refer to diagnostic hematology,
part two of this textbook) commonly known
as Abbé-type. This is a piece of equipment
in the microscopes that have built on-board
and is responsible for focusing the beam of
light into the slide and to the objectives and
then into the oculars. It is a significant part
in Köhler illumination and correction
(adjustment) of the light path. This is
composed of two linearly adjusted pieces of
lenses (condenser lenses) which can be
manipulated by means of condenser
focusing knob and condenser centering
adjustment knob. It has direct relationship
with the iris diaphragm and field diaphragm
where the light intensity and amount of light
will be controlled by opening or closing of
the iris diaphragm passing the light there
into the condenser.
Still in regards to microscope some terms
usually we hear related to the objectives and
these
are
achromatic objectives,
apochromatic objectives and planachromatic
objectives. Accordingly the achromatic
objectives refer to correction of chromatic
aberration and most of the brightfield
microscope have been made this way, it has
sharp focus at the center of the lens and lens
periphery are out of focus, also
apochromatic objectives means the
correction had been made for the chromatic
and spherical aberrations and finally the
planachromat objectives is the
objectives that lens in this is flat and it is
better for 40x and 100x magnifications: this
is because the lens is in focus and flat as
cited. It is more expensive in comparison to
other condenser lenses. In these lenses
chromatic aberration which is the short
blue light focus through glass or lens in
comparison to red band of light in the
spectrum of white light has been corrected
in these achromatic, apochromatic (with
spherical correction has also been done/vide
supra) and panachromat lens corrections.
The manufacturers of lenses make these
types of lenses to produce a high quality
lenses and to correct these lens defects. For
more information of light properties and
lenses such as refraction, retardation, angle
of incidence, refractive index, total
internal reflection, angle of refraction and
light waves characters such as amplitudes,
wavelength, frequency, coherent and noncoherent waves and other details of different
microscopies as well refer to J. D. Bancroft
et al. or other text books cited/Turgeon et al:
(physics of lens and the light waves)
(Bancroft et al).
Staining principles (Kennedy
et al
and Kedrnan et al)
Staining tissue is for studying cells and other
tissue components: this was introduced by
Leeuwenhoek in 1714. He was the
discoverer of microscope as well.
There are two types of dyes used in
histotechnology and histopathology. The
first are the natural dyes which are obtained
from biological sources, plants and seeds
and the second are the synthetic dyes,
artificially prepared by dye industry. Natural
dyes are further exemplified by saffron,
hematoxylin, orcein and mucicarmine or
584
commonly known as carmine. The
synthetic dyes example is aniline dye which
was the first synthetic dyes: other synthetic
dyes are derived from coal-tar, which are
the extracts and modifications of coal-tar.
Now-a-days biologic stain commission
(BSC) handles the upgrading and regulation
for the dye industry and the dye are
classified according to the color index
number (CI) which is a number unique to
each batch of dyes or stains manufactured to
avoid pitfalls of common names in dyeing
industry it consists of 5 digit number: i.e.
(32643); as with history of stains, it is
interesting to know that Henry Perkin’s is
renowned for synthesizing the first artificial
dye in 1856.
benzene. The Figure 2-3: shows the
Stains
Fig. 2-3: shows the benzene molecular
structure a predecessor molecule to different
coal-tar and subsequently different synthetic
dyes.
The most important natural and synthetic
dye
used
in
histotechnology
is
hematoxylin it is used in combination with
synthetic dye as eosin for tissue staining.
Other natural dyes also are used for the same
purpose in histotechnology as this is a
choice of the histotechnologist for dyeing
purposes. Additionally, Hematoxylin is
obtained
from
the
logwood
tree
“Hematoxylin
campechianum”:
hematoxylin in itself is not a dye and must
be oxidized to acidic hematein to become
a dye with its properties. Also carmine is
derived from cochneal: it is a red
crystalline purification product of this dye
(Gamble et al).
As with other dyes, natural dyes are
obtained from plant sources such as orcein
or saffron.
As said in relation to synthetic dyes, these
dyes are derived from or manufactured from
transformation of coal-tar derivatives.
Coal-tars are derivatives of hydrocarbon
molecular structure of benzene.
Benzene can transform into electrophilic
aromatic substitution as nitration,
sulfanation, halogenation and acylation.
These changes and transformations reflect
dyes
molecules
transformation
and
substitutions. Quinone or quinoid
compounds absorb light in the visible
spectrum however this molecule is
derivative of benzene (without any
chromophore and itself is a chromogen) and
benzene only absorb light at UV (ultraviolet)
region and has no color, nevertheless a
molecule such as trinitrobenzene has a
yellow color and is a chromophore: a
derivative of benzene, in this series, next
sequence also point that if trinitrobenzene
attaches to a auxochrome such as –OH
radical (hydroxyl radical), it will become
trinitrophenol or commonly known as picric
acid, a yellow dye used in histologic
staining.
585
The following terms are essential to
histotechnologist: 1) Chromophore, 2)
Chromogen and auxochrome, and 3)
Cationic and anionic dyes.
Chromophore is a substance in the dye
that can confer the dyeing properties to the
dye and to other substances, these
substances (group of atoms) can attach to
chromophore to able the chromphore to
become coloring and dyeable or absorb
light. The molecular structure that has
chromophore in it, is called chromogen:
this is the colored component of a dye: this
is although colored but has no dye property,
if there are more chromophore molecules in
the dye, it will have a denser and more
defined coloration or staining properties.
Therefore we have distinguished between
colored substance(s) and the dyes.
Chromophores are electron accepting groups
while auxochromes (another types of
molecules in the dye) are electron donating
groups. Nevertheless there are three
distinctive forms of molecules in dyeing and
staining: 1) Chromophore, 2) Auxochrome
and 3) the Chromogens. Combination of
chromogen and auxochrome enables the
production of a conjugated system as
chromophore. We have different types or
electron acceptors (chromophores): 1)
ethylene (C=C), 2) carbonyl (C=O), 3)
thiazol (C=S), 4) cyanic (C=N), 5) azo
(N=N), 6) nitroso (N=O), 7) nitro (NO2) and
8) quinoid.
Also we have three types or classes of dyes
1) Quinoid group, 2) Azo group, and the
3) Nitro group. In this respect, there are two
quinoid forms 1) paraquinoid, and 2)
orthoquinoid. The examples of these
(quinoid group) types of dyes are: basic
fuchsin, acid fuchsin, eosin, hematein
(hematoxylin), pararosaniline, aniline and
crystal violet. The next is the nitro (group)
dyes: picric acid and Martius yellow are of
these exemplary dyes. Azo dyes examples
are: orange G, chromophore 2R, panceau
3R, sudan IV, methyl red, congo red, oil
red O, luxol-fast-blue tartarazine, scarlet B
(biebrich scarlet) and metanil yellow
(Bancroft et al and Kiernan et al) and 3)
nitro dyes as trinitrophenyl.
Respectively
ionizing radicals in
auxochrome
enables
chromogen
(chromophore bearing molecule) to attach to
opposite charges, an cationic auxochrome
attaches to anionic (acidic) portion of the
tissue constituents such as nucleus, RNAs
etc. or vise-versa an anionic auxochrome
attaches to cationic (basic) portion of the
tissue constituents as cytoplasm.
Dyes are classified according to their basic
(cationic) or acidic (anionic) properties and
the way they attach to acidic or basic parts
of tissue components and these dye
properties are used in general or special
tissues staining.
Dye modifiers are molecules or chemicals
that alter the dye intensity such as methyl
groups (CH 3 ), or aryl group (C 6 H 5 ) or
ethyl group, etc. Dyes are called as
basophilic
(like
anionic
tissue
components) attaches to acidic tissue
components: i.e. nucleus and acidophilic
dyes (like basic components of tissues)
attaches to basic tissue components such as
cytoplasm. In neutral dyes both
components such as anionic and cationic
(ionizing components/auxochromes) are
present: example is Romanowsky stain
for neutrophils.
586
Amphoteric dyes act either as an acid (–)
or as a base (+). This depends on PH of the
surrounding tissues or the medium. Above
the IEP (isoelectric point) they act as an acid
and below as a base: i.e. as hematein in red
cells/erythrocytes and eosinophils.
Attention: It must be mentioned and noted
that PH indicators are colored but they are
not dyes they change color due to proton
transfer, but they do not confer dye
properties to other substances.
Basophilic tissues are divided into the
following tissues: they like cationic/basic
dyes and these tissues exemplified by
basophils, chromatin of the nucleus, DNA,
RNA, muscles, cartilages, and nucleic acid.
Acidophilic tissues like anionic/acidic
dyes and these tissues are exemplified by,
erythrocytes,
collagen,
eosinophils,
carboxylated, sulfated mucosubstances
and cell’s cytoplasm or etc.
In
tissue
staining
principles
and
mechanisms, there is coloration with dyes,
sliver impregnation methods, in situ
hybridization procedures, vital staining,
histochemical procedures, etc. here I will
explain only tissue coloration with
hematoxylin and esoin. For the rest a proper
textbook
in
histotechnology
and
histopathology should be consulted.
In dye coloration the most popular stains are
hematoxylin and eosin stains that apply to
the tissues. The steps involve include
regressive staining and progressive
staining: in regressive staining we
decolorize and differentiates the satins with
acid alcohol for differentiation (by acid
differentiation or mordant differentiation
and or by solvent differentiation) following
the initial stages of staining by washing with
Scott’s water or bluing agent to have a better
and brilliant staining however in progressive
staining the stain will be used in few step
procedures without decolorization and
differentiation. Hematoxylin is usually
performed by regressive staining (Gamble et
al & Kiernan et al).
Tissue staining
In direct staining of tissues, the use of
such stains as eosin for tissue accomplishes
with only one dye and the color varies with
the different shades of the same single color
with a simple single staining steps or
procedures, in this the staining/dye is in an
alcoholic or aqueous solution, while in
indirect staining the stain uses a mordant
to form tissue-mordant-dye complex to form
a lake. The mordant complex is not soluble
in the alcohol or aqueous solution, in
indirect staining the tissue is stained once
(primarily) and then it is differentiated
with some means (as mentioned/vide supra)
to remove the general staining of tissue
section except the component specifically
retained the stain.
In addition to staining mechanisms we
have different terms operating in this
laboratory specialty area thus we have
accentuators, accelerator, trapping agents
and argyrophil (Greek: love silver) and
argentaffin (Greek: neighboring silver)
reactions. As for some of them the short
definition are as follows: accentuators
increase the staining power of the dye; the
accelerators used in metallic impregnation
and are the same as accentuators. With
respect to argentaffin it means that it (this
type of cells) reduces ammoniacal sliver
nitrate solutions to metallic silver nitrite
rapidly and spontaneously. It does not need
the help from reducing substances as cellular
587
examples we have melanin (skin), and
entrochromaffin cells (i.e. as chromaffin
cells,
such
as
pheochromes
or
pheochromocytes,
enteroones
are
especially located in the intestine/ carcinoid
cells of the GIT/gastrointestinal epithelium
and cells of adrenal medulla are also
chromaffin cells and the related tumors, i.e.
pheochromocytomas can be used for
screening tissue sections for these tumors)
to reduce the ammoniacal sliver nitrate
solution to metallic silver nitrite, this is
accomplished by Masson Fontana method.
With respect to argerophil reaction, in this
method we do need the use of reducing
substances or light to reduce silver nitrate
solution to metallic silver (nitrite), i.e,
reticulin cells, the techniques is as in
Bielschowsky method: the reduction of
silver solution is done by the dye itself
which contains reducing substances.
In addition to factors that affect staining
quality are: PH of the medium, mordant,
temperature,
ionic
strength,
tissue
concentration and tissue density. Changes in
these parameter and factors obviously
affect the outcome of staining, for example
tissue density can trap the dye outside and to
some extend will not allow the dye to
penetrate the tissue and a reverse is true as
well. The temperature may affect staining in
such a way that it may over-stain or understain the selected tissue (s).
For concepts of metachromasia and its
types and leuco-compound refer to J.D.
Bancroft et al. or other sources available in
references. In short in metachromasia the
stain coloration by some tissue components
stains differently from original dye color (ation), example aniline dyes or toluidine
blue that changes color to dark pink when
dyeing the cartilage. In leuco-compound
the dye lose its dye properties due to
destruction or reduction of its chromophore
due to some tissue reduction characters and
there will be no coloration or dyeing, I put it
simply as this is a colorless compound
formed by reduction of the dye: these dyes
may retain their original dye color or
character by oxidation (using atmospheric
oxygen) or by other means.
The following discussion is how the
procedure of hematoxylin-eosin staining
can take place in tissue staining.
Usually when the H & E (hematoxylin and
eosin) staining is performed the
hemotaxylin stain would be done first
commonly in regressive form of staining
(vide supra) and then followed by eosin
staining. The hemotoxalyin stain as with
other dyes must be filtered before the
staining procedure and should be ripened
or oxidize to hematein to form a lake with
tissue and the mordant, called “tissuemordant-dye”. This ripening or oxidation
may be by air or atmospheric oxygen or
by chemical means (chemical oxidizers).
The hematoxylin must be in a light and
sunny place with complete exposure to room
air and sun’s light to ripen/oxidizes: this
may take 1-2 months as it is a slow process.
This may take up to 3 months to fully
oxidize, note that over-oxidation will
result into darker staining and even might
disqualified the stain from routine staining.
Mordant is used to attach the dye hematein
to the tissue and hematein on its own
cannot stain the tissues, therefore there is a
need of chemical or atmospheric oxidation
and thus mordant is to part/confer the
properties of tissue staining/dye to hematein.
The chemicals such as aluminum, ferric
ion, chromium and copper may acts as
mordant and forms cationic lakes with the
tissue components, also anionic lakes can be
formed by the action of mordants such as
588
phospho-tungstic acid and both (cationic
and anionic dyes) convey the staining
properties to the tissues. Figure 2-4 shows
the molecular structure of hematoxylin and
hematein with oxidation stage.
Fig.2-4: the molecular structures of
hematoxylin and hematien: with oxidation
sequence.
In this respect the combination of aluminum
and hematein will form hemalum or dyemordant-tissue complex (a lake)
commonly known as “aluminum lake”,
this binds to the phosphorly group of DNA
as a cationic lake and dyes the cellular
nucleus as blue. The hemalum stains tissues
a blue-lake. However the binding of ferric
salts to hematein forms “ferric lake”: (iron
hematoxylin/blue-black lake), may stain the
nucleus as blue-black as contrasted with
hemalum.
Once the oxidizer is acid such as mentioned
(phosphor-tungstic acid) it may form an
anionic lake. The principles of cationic
dyes and anionic dyes mentioned above
are applicable for hematoxylin and eosin
staining. In general hematoxylin stain is an
acid dye as it changes to cationic dye by
means hemalum or other cationic lakes,
although with phosphotungstic acid may
form anionic lake. In fact it is mostly used in
form of hermalum. In this respects eosin is
an acid stain used for cytoplasmic staining.
The physicochemical explorations of
staining are out of scope of this text. For
further explanation refer to the terminal
references.
There are different types of hematoxylin
stains with different chemicals and
components
namely
for
Alum
hematoxylin we have as: 1) Mayer’s
hematoxylin, 2) Harris’s hematoxylin, 3)
Cole’s
hematoxylin,
4)
Garrazzi’s
hematoxylin, 5) Gill’s hematoxylin; for
Iron/ferric hematoxylin we have: 1)
Weigert’s iron hematoxylin, 2) Heidenhain’s
iron hematoxylin, 3) Verhöeff’s iron
hematoxylin; and lastly the Tungsten
hematoxylin as: 1) Phophotungstic Acid
Hematoxylin
(PTAH);
and
for
Molybdenum hematoxylin, we have 1)
Phosphomolybdic acid stain. For routine H
& E (Hematoxylin & Eosin) staining we use
Alum hematoxylin sloutions.
Attention: If the tap water is not alkaline
use Scott’s bluing substitutes as bluing
agents and all stains/dyes must be filtered
before application in staining methods, this
will clear impurities and contaminants at one
time.
Staining of H and E may be accomplished
by regressive or progressive staining. In
progressive all basophilic tissues such as
nuclei are blue in color as stain with
hematoxylin, at next stage if water is not
alkaline used bluing agent (such as
Scott’s), and then wash and check
microscopically for proper staining of the
tissue elements, proceeds with buckets of
eosin stain: once stained with eosin rinse
with tap water and dehydrate, clear in
589
xylene and mount the slide with tissue and
coverslip, the cytoplasm looks pink with
eosin. The regressive is the most popular
and common in comparison with
progressive. The staining proceeds with
the same sequence of staining with
progressive staining only when hematoxylin
stain is applied, it will be followed by
microscopic differentiation with acidalcohol solution (decolorizer). The rest
remains with eosin steps. Follow the
incumbent laboratory schedules and timing
for H & E staining protocols, the following
figure (Fig. 2-5) is the molecular structure of
eosin as a histologic dye (Kiernan et al)
strength, and 4) Fixation quality. As an
example PH factor may affects staining
quality by using dyes such as amphoteric
dyes, or a leuco-compound and tissue
metachromasy, while other stains may
need an act of staining at an optimal and
certain PH to result in an optimum quality as
clarity; stain purity/or impurities and
brilliant staining outcome. The type of
fixation may as well affect the staining
quality this is the same for dehydration
and clearing which all have critical effects
on staining properties.
Chapter Three (3)
A
ll the following stains reagents and
methods/procedures have been
taken from the following original on
line Web sources (URL):
(http://www.histologycourse.com/Pigment%
20%26%20Mineral%20Staining.pdf)
Staining of mucins
(Gamble et al,
Lamar et al & Bancroft et al.)
Fig. 2-5: the molecular structure of eosin
Other stains/dyes such as phloxine B,
erythrosine (tetraiodofluorescein), eosin
Y (tetrabromofluorescein), eosin
B,
biebrich scarlet (scarlet B) and orange G
are used in histotech lab for the staining
purposes of cytoplasm. Lastly there are
factors that affect staining quality by
optimization of the staining process these
enumerates namely as: 1) Tissue
components, 2) PH of the mordant, 3) Ionic
Mucin or mucins (which indicate a more
complex nature of mucin) are carbohydrates
containing
macromolecules
such
as
glycogen, and mucosubstances. Other
glycogen and carbohydrates substances
(polysaccharides) found in the in the body
and typed as mucins include mucins in
cartilages known as chondroitin sulfate,
lyoglycogens and desmoglycogen all are
various types of mucins, they can be found
in epithelial cell lining of intestinal tract, or
respiratory and reproductive tract and or in
pancreas and breast. In this respect the
proteoglycons are not mucins and will not
590
discuss further in this section. The purpose
of tissue demonstration of such substances is
indicated in tumor and glycogenoses
detection and study in such tissues, there are
several tissues staining techniques used in
the demonstration of mucins. As we can
name
there
are
staining
procedures/techniques such as alcian blue,
PAS
(Periodic
Acid
Schiff),
mucicarmine/carmine, and colloidal iron
techniques.
There is specific amino acid core or protein
core in mucins with tandem repeats in its
molecular structures which these repeats
depend on the size and variation in amino
acids of the mucins molecule. These are
typed as MUC 1 (with distribution of
episialin in ovaries, pancreas and breast),
MUC 2 (with larger distribution in large and
small intestines), MUC 3 (within Jejunum)
and MUC 4 (distribution within transbronchial mucosa), etc. MUC (Mucin)
genes codes for these different genetically
distinct
forms
of
glycoproteins/mucosubstances. The staining
property and histochemical reactivity
can be applied to carbohydrate moiety of the
molecules not the protein portion or the
core.
There are two types of mucins, one is acid
mucins: its divisions are such as strongly
sulfated (as in connective tissues and
epithelial
cells);
weakly
sulfated,
carboxylated sialomucin and sulfated
sialomucin (such as in hyaluronic acid).
The second one is neutral mucins with no
subdivisions.
Mucins special stains have cationic charged
group (vide supra) in solutions at specific
PH. Stains such as alcian blue and
muncicarmine has cationic properties and
joins/attach to anionic portion of anionic
carboxylated or sulfated polysaccharide
chain of mucins molecules in the tissues by
means of electrostatic forces, thus conveying
the coloration properties. Figure 3-1,
indicates Alican blue/PAS staining of
rat colon, note the coloration of nucleus and
cytoplasm with dark blue stains for mucins.
Combination of alcian-blue and PAS can
differentiate between neutral mucins and
acid mucins. In most protocols we stain
with standard alcian-blue and subsequently
stain with Periodic Acid Schiff (PAS).
This subsequent staining with PAS stains
neutral mucins are bright magenta. The
tissues and cells that contain both mucins
color dark blue or purple coloration.
Fig. 3-1: the alcian-blue/PAS technique in
staining rat colon: showing blue and
magenta color of nucleus and cytoplasm
respectively: note the accumulation of blue
color at the periphery of the epithelial
glandular crypts of Lieberkühn.
Staining of connective tissue
(Lamar et al)
591
Connective tissues provide maintenance and
support of other types of tissues in the
body. There are different types of
connective tissues. It connects and
support as a matrix of other cells and tissues
as per se. These include osseous bone (as
will be covered in bone staining section),
and organs such as pancreas, heart and
kidney that need support by connective
tissues, these connective tissue substances
and components include, collagens, elastic
fibers (which imparts strength to the
connective tissue), reticulin (reticular
fibers). The cells in the connective tissues
are mast cells, histocytes, fibroblasts,
mesechymal cells, plasma cells, blood cells,
adipose (adipocytes) and fat tissues and
marcrophages (as histocytes etc.).
The best tissue stains for connective tissue
fibers as collagen may consist of Masson,
Gomori and van Gieson techniques. The
reticulin fiber as a sort of collagen may be
detected by means of argyrophilic
reaction/techniques as mentioned earlier in
staining methods. For screening of elastic
fibers as a kind of connective tissue which
gives strength and elasticity to the tissues
are Verhoeff, van Gieson, Weigert and
Gomori’s stains, as well H & E stains are
the routine staining techniques for these
fibers.
Accordingly collagen is the most common
connective tissues fibers. It is normally
stained with classification such as collagen
type I, II, II, and etc. These may be named
as Collagen type I would be the most
common collagen, type II is the articular
cartilage and type III collagen would be of
granular tissues, type IV is the basal
lamina, etc. The most popular stain for
collagen is the Masson trichrome stain. The
reaction of this stain is based on the three
dyes as: 1) Weigert’s hematoxylin, 2)
Beibrich Scarlet/acid fuchsin, and 3)
aniline blue. In this reaction nuclei are
black, keratin, cytoplasm and muscle fibers
are red and collagen and mucous is blue.
The following are the Mason trichrome
stains reagents and procedures and Figure
3-2 shows the staining properties of
Masson‘s trichrome in a tissue: All the
procedure and reagent protocol presented in
this part of the text is taken from Web
address referred in the references.
Masson’s Trichrome Stain
Purpose of the stain: To differentiate
between collagen and smooth muscle in
tumors and to identify increases in
collagenous tissue in diseases such as
cirrhosis of the liver.
Principle of the stain: This reaction is
based on a three dye stain.
Fixatives: Bouin’s solution is preferred, but
10% Neutral Buffered Formalin (NBF)
may be used.
Sectioning: Paraffin sections at 4-5 microns.
Controls: Every tissue has an internal
control, but uterus, small intestine,
appendix or fallopian tube will be good
material.
Reagents:
1. Bouin’s Solution: Picric acid saturated
formaldehyde & Glacial acetic acid.
2. Weigert’s Iron Hematoxylin: Solution A:
Hematoxylin & 95% Alcohol; & Solution B:
29% Ferric chloride, Distilled water &
glacial acetic acid. Mix equal parts of
solution A & B.
3. Biebrich Scarlet-Acid Fuchsin
Solution: 1% Biebrich Scarlet, 1% Acid
Fuchsin & Glacial acetic acid.
592
4. Phosphotungstic/Phosphomolybdic Acid
solution:
Phosphomolybdic
acid,
Phosphotungstic acid & distilled water.
5. Aniline Blue solution: Aniline blue dye,
Glacial acetic acid & distilled water.
6. 1% Acetic acid solution: Glacial acetic
acid & distilled water.
Procedure:
1. Deparaffinize and hydrate slides to
water.
2. Rinse slides well in water.
3. Place sections in Bouin’s solution for 1
hour at 56 degrees Celsius. This step is
where we the tissue is be mordanted.
4. Rinse slides in running tap water. This
step is necessary for the total removal of the
yellow coloring on the sections.
5.
Stain
sections
in
Weigert’s
hematoxylin for 10 minutes. This step is
where we are staining the entire nucleus.
6. Rinse slides in running water for 10
minutes. This step helps with the removal of
excess Weigert’s Hematoxylin Stain.
7. Stain slides in Biebrich Scarlet-acid
fuchsin for 2 minutes. This step will stain
the cytoplasm of the tissue and other tissue
elements.
8. Rinse slides in running tap water to
remove any excess staining.
9.
Place
slides
in
the
Phosphotungstic/Phosphomolybdic
acid solution for 10 to 15 minutes. This
step removes any staining that has occurred
in areas not cytoplasmic.
10. Stain slides in Aniline blue solution
for 5 minutes. This is recommended for
small amount of collagen. For large amount
of collagen Light green counterstain is
recommended. This step is the collagen
staining step.
11. Place slides in 1% acetic acid solution
for 3 to 5 minutes. . This is a differentiating
step.
12. Dehydrate slide with 95% alcohol, 100%
alcohol, clear in xylene and coverslip with
a resinous mounting media
Results:
Nuclei…..…………………………………
…………..…….. Black
Cytoplasm,
keratin,
muscle
fibers…………………………... Red
Collagen
and
Mucous………………………..……………
….Blue
Fig. 3-2: shows the nuclei black, cytoplasm
red and collagen blue by Mason trichrome
stain in a tissue, it stains fibers in heart,
kidneys, lungs and other connective tissue
bearing organs.
Elastic fibers are produced by fibroblasts
and smooth muscle cells of connective
tissues of arteries it expands and stretches to
give flexibility to the organs. These are
bundles of proteins. The chosen stain for the
elastic fibers are Verhöeff elastic stain,
aldehyde fuchsin elastic stain. The Verhöeff
staining procedure and reagent are presented
in the following; Figure 3-3 shows the
Verhöeff staining of elastic fibers.
Verhöeff’s Elastic Fiber Stain
593
Purpose of the stain: Demonstration of
elastic fibers in target tissue.
Principle of the stain: The tissue is over
stain with a solution of hematoxylinferric chloride - iodine solution. Ferric
chloride and iodine serve as mordant, but
they also have an oxidizing function in
helping the conversion of hematoxylin to
hematein.
Fixatives: Any well-fixed tissue may be
used, 10% Neutral buffered formalin or
Zenker’s is preferred.
Controls: Aorta embedded on edge or a
cross section of a large artery.
1. Lugol’s iodine: iodine, potassium
chloride and distilled water.
2. 10% Ferric chloride: Ferric chloride &
Distilled water no
3.
Alcoholic Hematoxylin, 5%:
Hematoxylin & 95% Ethanol
4. Verhoeff’s Elastic stain: 5% alcoholic
hematoxylin, 10% Formal Lugol’s Iodine.
Van Gieson Solution
6. 5% Sodium thiosulfate: Sodium
thiosulfate & distilled water.
:
Procedure
1. Deparaffinize and hydrate slide to water
2. Place sections in Verhoeff’s Elastic stain
for 1 hour, this solution must be prepared
fresh and in the order given, otherwise poor
staining may result. .
3. Wash slides in 2 changes of distilled
water. This step will remove excess
Verhoeff’s Elastic stain.
4. Differentiate section in 2% Ferric chloride
until elastic fibers are distinctive and the
background is colorless or light gray. If
sections are differentiated too far, re-stain
in the Verhoeff’s elastic stain. This step is a
critical step, since we are dealing with the
differentiation of elastic fibers.
5. Rinse slides in distilled water.
6. Place sections in Sodium Thiosulfate
for 1 minute. This step will remove any nonspecific staining from the tissue section.
7. Wash slides in running tap water for 5
minutes.
8. Counterstain slides in van Gieson
solution for 1 minute. This step will allow
for
the staining of collagen, muscle and
cytoplasm. Differentiate slides in 95%
alcohol.
9. Differentiate slides in 95% alcohol.
10. Dehydrate slide 100% alcohol, clear in
xylene and coverslipe in rezinous
mounting media.
Results:
Elastic
fibers..............................................................
........Blue-black to black
Nuclei.............................................................
...................Blue to black
Collagen.......................................................
.....................Red
Other
tissue
elements........................................................
Yellow
594
Fig. 3-3: the Veroeff-VanGieson stains of
elastic fibers of the Aorta, for coloration
results refer to text.
Reticular fibers technique Figure 3-4
shows staining with silver nitrate staining of
reticular fibers:
Silver Techniques for Reticular
Fibers
1. Oxidation: Changes of the glycol group to
the aldehyde groups.
- Phosphomolybdic acid
- Potassium permanganate
- Periodic acid
2. Sensitization: Usually is a metallic
impregnation step. Impregnation is the
deposition of metallic salts on or around, but
not in, the tissue element to be
demonstrated. The exact chemical reaction
is not understood. The sensitizing metal is
then replaced by silver.
- Uranyl nitrate
-Ferric ammonium sulfate
-Dilute solution of silver
nitrate
3. Silver Impregnation: Involves treating
tissue with an ammoniacal or diamine silver
complex.
4. Reduction: The silver is reduced into a
visible metallic state.
- Formaldehyde
5. Toning: Term used when bound metallic
silver is treated and the color of the
impregnated component changes from
brown to black. The metallic silver is being
replaced by metallic gold.
Gold
Chloride
(toning agent)
6. Unreduced silver: This step prevents any
non-specific bound silver remaining in the
section from being reduced by a later
exposure to light.
- Sodium Thiosulfate
(Hypo)
7. Counterstain: May or may not be used, is
up to the laboratory procedures.
- Nuclear Fast Red (If
counterstain is used)
Fig. 3-4: silver nitrate staining in rat
pancreas: the stain is used for
neuroendocrine,
paracrine
and
neurofibrillary tangles in CNS, in response
and reactivity to chromaffin cells in these
tissues, also positive in entrochromaffin
595
cells
of
GIT
(gasrrointestinal
pheochromaffin cells): it is an excellent
detection tool in tumors of neurofibrillary
and
endocrine
cells
such
as
pheochromocytomas.
Gomori Stain for Reticular Fibers
Purpose of the stain: Demonstration of
reticular fibers in tissue sections. A change
of normal pattern as seen in some liver
diseases, is also an important diagnostic
finding.
Principle of the stain: The tissue will be
oxidized converting the sugar group to
aldehyde followed by a sensitizing step for
metallic salts. Impregnation of with silver
will follow, then it will be reduced to a
metallic visible state by formaldehyde.
Gold chloride is applied as a toner which
will change the bound silver from a brown
color to a black color. A hypo solution
will follow in order to remove any
unreduced silver, and if desired a
counterstain is applied.
Fixatives: 10% Neutral buffered formalin is
preferred.
Sectioning: Cut paraffin sections from 4
to 5 microns.
Controls: Liver is a very good control
tissue.
Reagents:
1. 10% Silver nitrate: silver nitrate &
distilled water.
2. Potassium hydroxide solution: potassium
hydroxide & distilled water.
3. Ammoniacal silver solution: 10%
silver nitrate & potassium hydroxide.
Concentrate ammonium hydroxide is
added drop by drop until it becomes slightly
cloudy and then clear again.
4.
0.5%
Potassium
permanganate:
potassium permanganate & distilled
water.
5. 2% potassium metabisulfite: Potassium
metabisulfite & distilled water.
6. 2% ferric ammonium sulfate: Ferric
ammonium sulfate & distilled water.
7. Formalin solution: Formaldehyde, 37 to
40% & distilled water.
8. 0.2% Gold chloride: Gold chloride and
distilled water.
9. 2% Sodium thiosulfate: Sodium
thiosulfate & distilled water.
10. Nuclear Fast Red
Procedure:
1. Deparaffinize and hydrate slides to water.
2. Place slides in 0.5% potassium
permanganate solution for 1 minute. This
step is changing the glycol group to
aldehyde group.
3. Rinse slides in tap water for 2 minutes.
4. Place slides in 2% potassium
metabisulfite for 1 minute. This step will
remove the purple color from the tissue
section.
5. Rinse slides in tap water for 2 minutes.
6. Place section in 2% ferric ammonium
sulfate for 1 minute. This is the
sensitization step.
7. Rinse slides in tap water for 2 minutes.
Followed by two changes of distilled
water.
8. Place section in the Ammoniacal silver
for 1 minute. This is the impregnation step.
9. Rinse slides in distilled water for 20
seconds.
10. Place slides in formalin solution for 3
minutes. This is the reduction step.
11. Rinse slides in tap water for 3 minutes.
596
12. Place slides in 0.2% gold chloride
solution for 10 minutes. This is the toning
step.
13. Rinse slides in distilled water.
14.
Place
slides
in
potassium
metabisulfite for 1 minute.
15. Place slides in 2% sodium thiosulfate.
This is the step where the unreduced silver is
removed.
16. Rinse slides in tap water for 2 minutes.
17. Counterstain if desired with Nuclear
Fast Red for 5 minutes.
18. Rinse well in tap water.
19. Dehydrate slide with 95% alcohol, 100%
alcohol, clear in xylene and coverslip with a
resinous mounting media.
Results:
Reticulin
fibers.........................................................Bl
ack
Collagen.........................................................
...........Taupe
PTAH for Cross-Striations and
Fibrin
Purpose of the stain: For the demonstration
of cross-striations and fibrin. Crossstriations are a diagnosis feature of
rhabdomyosarcomas or tumors arising from
striated muscles figure 3-5, shows PTAH
staining of muscle fibers..
Principle of the stain: This stain has been
referred to as a polychrome stain because
one solution can give two major colors.
Fixatives: Zenker’s solution is preferred,
but 10% NBF can be used.
Sectioning: Cut paraffin sections from 4 to
6 microns.
Controls: Skeletal or cardiac muscle for
cross-striations or a section with fibrin for
fibrin demonstration, or cerebral cortex for
glial fibers demonstration.
Reagents:
1.
PTAH
solution:
Hematoxylin,
Phosphotungstic acid, distilled water.
2. Gram Iodine: Iodine, Potassium Iodide &
distilled water.
3. 5% Sodium thiosulfate & distilled water.
4. 0.25% Potassium Permanganate:
potassium permanganate & distilled water.
5. 5% Oxalic acid: oxalic acid & distilled
water.
Procedure:
1. Deparaffinize and hydrate slides to water.
2. If tissues are formalin fixed, mordant in
Zenker’s solution containing 5% acetic acid
overnight at room temp.
3. Place slides in tap water.
4. Place slides in Gram Iodine for 15
minutes.
5. Rinse slides in tap water.
6. Place slides in 5% sodium thiosulfate
solution for 3 minutes.
7. Rinse slides in tap water for 10 minutes.
8. Place sections in 0.25% potassium
permanganate for 5 minutes.
9. Rinse slides in tap water.
10. Place slides in 5% Oxalic acid for 1
minute.
11. Wash slides in running tap water for 10
minutes.
12. Stain slides in PTAH solution overnight
at room temperature.
13. Dehydrate rapidly through two changes
of 95% alcohol, 100% alcohol, clear in
xylene and coverslip.
Results:
Cross
striations
and
Fibrin.......................................................Blu
e
Nuclei.............................................................
.........................Blue
597
Collagen.........................................................
.........................Red Brown
Fig.3-5: the striated muscles (skeletal
muscles) appear blue with red cells and the
perivascular collagen appears reddish brown
in PTAH staining for distinction of
rabdomyosarcoma: a malignant tumor of
muscle fibers.
Jones Method (Periodic
Methenamine Silver)
Acid
Purpose of the stain: This procedure best
delineates
the
glomerular
basement
membrane Fig. 3-6, shows methenamine
sliver stain in tissues of glomerular
basement membrane.
Principle of the stain: Methenamine silver
demonstrates the carbohydrate component of
basement membrane by oxidizing the
carbohydrate to an aldehyde. The tissue
will be oxidized converting the sugar group
to aldehyde followed by a sensitizing step
for metallic salts. Impregnation of with
silver will follow. Gold chloride is applied
as a toner which will change the bound
silver from a brown color to a black color. A
hypo solution will follow in order to remove
any unreduced silver, and light green is
applied as a counterstain.
Fixatives: 10% Neutral buffered formalin is
preferred.
Sectioning: Cut paraffin sections from 2
microns.
Controls: Kidney has an internal control.
Reagents:
1. Stock Methenamine Silver: 3%
methenamine solution & 5% silver solution.
2. 5% Borax solution: Sodium borate &
distilled water.
3. Working Silver: Stock methenamine
silver, Borax solution and distilled water.
4. 1% Periodic acid solution: Periodic acid
& distilled water.
5. 0.02% Gold Chloride Solution: Gold
chloride & distilled water.
6. Light Green Solution: Stock light green
solution & distilled water.
Procedure:
1. Deparaffinize and hydrate slides to
water.
2. Place slides in 1% Periodic acid
solution for 15 minutes. This is the
oxidizing step where is changing the glycol
group to aldehyde group.
3. Rinse slides in tap water for 2 minutes.
4. Place section in the Methenamine silver
for 15 to 20 minute. This is the impregnation
step.
5. Rinse slides in heated distilled water.
6. Place slides in 0.2% gold chloride
solution for 2 minutes. This is the toning
step.
7. Rinse slides in distilled water.
8. Place slides in 2% sodium thiosulfate
for 1 minute. This is the step where the
unreduced silver is removed.
9. Rinse slides in tap water.
10. Counterstain in light green solution for
1 1/2 minutes. Dehydrate slide with 95%
alcohol, 100% alcohol, clear in xylene and
coverslip with a resinous mounting media.
598
Results:
Basement
Membranes.................................................
........Black
Background....................................................
.....................Green
Fig. 3-6: methenamine sliver stain (MST) of
glomerular basement membrane in a case of
renal failure: indicates proliferative,
necrotizing sclerosis of basement membrane
with vasculitis and extravasation of red cells
in the bowmen’s capsule: note the black
basement membrane and bluish green of
background.
Oil Red O Method for Neutral Fats
Purpose of the stain: Demonstration of
neutral lipids in frozen sections, Figure 37, presents fat cells demonstration with oli
red O staining techniques.
Principle of the stain: This is a physical
stain and the dye used must be more soluble
in the tissue lipid than in the solvent in
which it is dissolved. Must not be water
soluble. Must be strongly colored, and must
act with tissue constituents only by solution.
Fixatives: 10% NBF or calcium formol,
because of lipid dissolving abilities, no
alcoholic fixatives should be used.
Sectioning: Cut frozen sections at 10
microns.
Controls: Most tissue contains fats,
normally no control is needed.
Reagents:
1. Oil Red O Stock solution: Oil Red O,
Isopropanol, 98%.
2. Oil red O Working solution: Oil Red O
Stock solution & Distilled water. Mix well
and let stand for 10 minutes before using.
Procedure:
1. Cut frozen sections and fix in 40%
formaldehyde for 1 minute, and wash well in
tap water. Blot off excess water. If the
tissue has been previously fixed, then there
is no need of fixation.
2. Stain slides in Working Oil Red O
solution for 10 minutes.
3. Wash sections in tap water.
4. Stain slides in Harris’ hematoxylin with
acetic acid for 1 minute. This step will stain
all the nuclear details of the tissue.
5. Wash slides in running tap water for
several minutes.
6. Blue slides in ammonium water.
7. Wash slides in tap water.
8. Mount sections with an aqueous mounting
medium.
9. Seal edges with fingernail polish.
Results:
Fat
........................................................................
.Intense Red
Nuclei.............................................................
........Blue
599
Pigments are coloring agents that may
deposit in cells or tissues as cytoplasmic
and they are granular in nature. As above,
these (pigments) are divided into artifacts,
hematogeneous and non-hematogeneous
in origin.
Artifacts are artifactual debris and these are
deposited in the cells and tissues during
dyeing or other chemical manipulation of
tissues such as mercury deposits,
formaldehyde pigments and chrome
pigments. Exogeneous pigments are made
outside of the body and due to
contamination or processes they appear
internally, i.e are carbon, asbestos fibers,
and tattoo
pigments.
Endogenous
pigments being mentioned already and
they are originated internally.
.
Fig. 3-7: reddish neutral fat stained with oil
red O, note the blue nuclei in the matrix.
Staining of pigments
(Bacroft et
al)
Endogenous
pigments
are
either
hematogeneous or non-hematogenous.
Hematogeneous is divided to hemoglobin
with hem (as the pigment part) and globin
portion as protein portion (in amino acid
pool).
Hem is further divided to iron and bile
pigment. In here bile pigment is biliverdin
(with bilirubin its further product.). In
addition iron can be reused in the
hemoglobin or be broken down to
hemosiderin
Endogenous hematogeneous pigments such
as hemoglobin is stained with eosin stain, or
hemosiderin as found in conditions such as
hemosiderosis would be stained with
Perl’s Prussian Blue stain (PPB). Also bile
pigment, biliverdin and bilirubin found in
jaundice may be stained with Hall’s stain for
bile pigment with Fouchet reaction.
In consideration of endogenous nonhematogeneous pigments we have melanin
as a pigment or chemical bound to proteins
and lipofuchsin as for lipid pigments.
Regarding the melanin it is found in hair
shafts, skin, retina, iris and certain parts of
CNS (central nervous system) and is a
brown to black pigment. It is stained with
bleaching
and
10%
potassium
permanganate. With regards to lipofuchsin
as said we have wear and tear pigments as
lipofuchsin and are found in heart, liver and
neurons. To detect lipofuchsins and lipidic
pigments we utilize Oil Red O or Sudan
black B (SBB).
600
For endogenous deposits we have urea
deposited in tissues as in gout tophi and
Gout. Other ions such as cupric, ferrous and
ferric, calcium, phosphate and carbonate
may deposit in tissues and interfere with
normal interpretation of the histopathology
slide(s) these may be normal constituents of
bodily tissues electrolytes or so. Whereas
minerals such as silver, lead, copper and
gold are heavy metallic deposits and are
always
pathologic/heavy
metal
toxicity/poisoning. The other most known
pigments of cytoplasmic granular in origin
include ‘C” cells of thyroid, pancreatic
endocrine cells and entrochromaffin cells
of intestines along with adrenal chromaffin
cells and neuroendocrine cells of nervous
system and endocrine system. These are
being demonstrated
by means
of
argyrophils (need a reducer) and
argentaffin techniques as mentioned in
pervious sections (vide supra). Some of the
special stains reagents and procedures are
included in here:
Prussian Blue Stain for Ferric Ion
(Fe+3)
Purpose: The detection of ferric iron in
tissue. Found normally in spleen and bone
marrow.
Principle: Detects the loosely bound protein
complexes (As in hemosiderin). Strong
bound iron will not react. Sections are
treated with an acidic solution of potassium
ferrocyanide and any ferric ion present will
react in a bright blue pigment called
Prussian Blue.
Fixative: Alcohol or 10% Neutral buffered
formalin.
Technique: 4 to 5 μm paraffin sections.
Control: a section containing ferric iron
must be used.
Reagents: 2% Potassium Ferrocyanide:
Potassium ferrocyanide & distilled water
2%
Hydrochloric
acid
Solution:
Concentrated hydrochloric acid & distilled
water
Nuclear Fast Red solution: Nuclear fast Red,
Aluminum sulfate, distilled water & Thymol
Procedure:
1. Deparaffinize and hydrate slides to water
2. Place slides in a freshly prepared solution
of a 50:50 mixture of 2% potassium
ferrocyanide & 2% hydrochloric acid for 20
o
minutes at 60 C.
3. Wash slides thoroughly in water.
4. Counterstain slides in Nuclear fast Red
solution for 5 minutes.
5. Rinse slides in running tap water for 1
minute.
6. Dehydrate sections in 95% alcohol and
two changes of absolute alcohol.
7. Clear slides in three changes of xylene
and mount in a synthetic resin mounting
media.
Results:
Nuclei
&
hemofuchsin
………………………………………………
…...…Bright Red
Hemosiderin
(iron)………………………………………
……………….….Blue
Background…………………………………
………………………….…….Pink
Following figure 3-8 is the staining pattern
of Prussian blue of bone marrow deposits
of iron.
601
ferricyanide, pH to 2.4 with 1N hydrochloric
acid
Mayer’s Mucicarmine Solution
Metanil Yellow: Metanil yellow, distilled
water & glacial acetic acid
Procedure:
Fig. 3-8: Perl’s Prussian Blue (PPB) staining
of bone marrow for iron deposits, blue
deposits indicates iron deposits. Note the
fat/cell ratio.
Schmorl’s
Technique
Reducing Substances
for
Purpose: The detection of ferrous iron in
tissue.
Principle: Detects the ferrous iron in tissue.
Sections are treated with an acidic solution
of potassium ferricyanide and any
ferrous iron present will react to form an
insoluble bright blue pigment called
Turnbull’s blue (ferrous ferricyanide).
Fixative:
10%
Neutral buffered
formalin.
Technique: 4 to 5 μm paraffin sections.
Control: a section containing melanin or
argentaffin granules.
Reagents:
1% Stock ferric chloride: Ferric chloride &
distilled water
0.1% Potassium Ferricyanide: Potassium
ferricyanide & distilled water
Ferric chloride-Potassium ferrocyanide: 1%
ferric chloride & 0.1% Potassium
1. Deparaffinize and hydrate slides to
water.
2. Place slides in a freshly prepared ferric
chloride - potassium ferricyanide solution
and stain for 3 changes of 7 minutes each.
3. Rinse sections in distilled water.
4. Stain slides in working Mayer’s
Mucicarmine for 1 hour.
5. Rinse slides rapidly in distilled water.
6. Counterstain slides in Metanil yellow
for a few seconds.
7. Rinse slides rapidly in distilled water.
8. Dehydrate sections in 95% alcohol and
two changes of absolute alcohol.
9. Clear slides in three changes of xylene
and mount in a synthetic resin mounting
media.
Results:
Reducing
Substances.………………………………
………..………………Blue-green
Goblet
Cells,
mucin………………………………………
…………….……Rose
Background…………………………………
……………………………….Yellow-green
Following figure (3-9) is for Schmorl’s
technique of tissue for reducing substances.
602
the DNA bonds at 60°C then the exposed
aldehyde is demonstrated with PSA (Schiff
reagent) .
Fontana Masson for Melanin and
Argentaffin granules
Fig.3-9: indicates chromaffin cells in adrenal
medulla of mice stained with Schmorl’s
potassium ferricyanide techniques.
It is out of the scope of this text to present
all stains reagent and procedure with
regard to pigments and other tissues
although I will name the stain procedures for
each pigment in here to know the detail
techniques and staining protocol refer to the
terminal references.
In addition to the above we use FontanaMasson technique for argentaffin
substances, cells (tissues), for argyrophil
cells (need reducing substances) we apply
Grimelius’
argyrophil
staining
technique. Also for the same substance
detection, Churukian-Schenk argyrophil
technique may be added to this staining
protocol. In concern of urates the ideal
staining
method
is
Gomori’s
methenamine silver stain. As mentioned
for bile (salts) staining Hall’s techniques are
available (also modified Fouchet/reaction
staining technique is the classical staining
techniques for liver bile salts/pigments). For
detection of calcium Von Kossa and
Alizarin red S stains are used in routine
staining of calcium deposits. And lastly
rhodamine stain is used for copper with
Feulgen reaction for DNA (deoxyribose)
staining: in this acid is used to breakdown
Purpose: Demonstration of argentaffin
substances such as melanin, argentaffin
granules of carcinoid tumors, and some
neurosecretory granules.
Principle: Some processes are argentaffin
meaning they have the ability to bind silver
from a silver solution and to reduce it to the
visible metallic silver without the need for a
separate reducing agent.
Fixative: 10% Neutral buffered formalin
(NBF), alcoholic fixative should be avoided
since it tends to dissolves the argentaffin
granules.
Technique: 4 to 5 μm paraffin sections.
Control: a section of skin for melanin,
appendix or small intestine for argentaffin
granules.
Reagents:
10% Silver Nitrate
Fontana Silver Solution: 10% Silver Nitrate
& Ammonium hydroxide
Gold Chloride (toning agent)
Sodium Thiosulfate
Nuclear Fast Red
Procedure:
1. Deparaffinize and hydrate slides to water.
2. Place slides in silver nitrate solution in
water bath at 56 degrees. For 1 hour.
3. Rinse sections in distilled water.
4. Immerse slides in gold chloride for 10
minutes.
5. Rinse slides rapidly in distilled water.
6. Place slides in sodium thiosulfate for 5
minutes.
7. Rinse slides in distilled water.
8. Counterstain slides in Nuclear fast Red
for 5 minutes.
603
9. Wash slides for at least 1 minute in
distilled water.
10. Dehydrate sections in 95% alcohol and
two changes of absolute alcohol
11. Clear slides in three changes of xylene
and mount in a synthetic resin mounting
media.
Results:
Melanin
.………………………….…………………
……….............................…Black
Argentaffin
granules……………………………………
…………………………Black
Nuclei……………………………….............
....………………………………….Pink
Figure 3-10 shows staining of tissue with
Masson-Fontana technique for melanin and
argentaffin cells.
Fig. 3-10: Masson-Fontana staining of
melanin in epithelium for demonstration of
argentaffin cells in the tissue section: note
that the detections are used for diagnosis of
chromaffin cell carcinoids and cancer of the
said tissues such as pheochromocytoma of
adrenal
and
skin/intestine
neuroendocrine glands.
and
Grimelius’ Argyrophil Stain
Purpose: Demonstration of argyrophil
granules in neurosecretory tumors.
Principle: Some processes are argyrophilic
meaning they need a reducing agent in order
to bring the silver to a visible metallic form.
Fixative: 10% Neutral buffered formalin
(NBF).
Technique: 4 to 5 μm paraffin sections
Control: an argyrophilic-positive carcinoid
tumor is preferred, but a section of small
intestine can be used. Glassware should be
chemically cleaned.
Reagents:
1% Silver Nitrate: Silver nitrate & Distilled
water
0.2M Acetic acid: Glacial Acetic acid &
Distilled water
0.2M Sodium Acetate: Sodium acetate &
Distilled water
Working Silver Solution: Acetic acid-acetate
Buffer, 1% Silver nitrate & Distilled water
Reducing Solution: Hydroquinone, Sodium
sulfite & Distilled water
Nuclear Fast Red: Nuclear Fast Red,
Distilled water & Thymol
Procedure:
1. Deparaffinize and hydrate slides to water.
2. Place slides in working silver nitrate
solution in water bath at 60 degrees for 1
hour.
3. Drain slides briefly.
4. Place slides in freshly prepared reducing
solution preheated to 40 - 50 degrees Celsius
for 1 minute.
5. Rinse slides well in distilled water.
6. Repeat step 2 for 10 minutes.
7. Drain slides briefly and place in reducing
solution again for 1 minute.
8. Rinse slides in distilled water for 2
minutes.
604
9. Counterstain slides in Nuclear Fast Red
solution for 5 minutes.
10. Dehydrate sections in 95% alcohol and
two changes of absolute alcohol
11. Clear slides in three changes of xylene
and mount in a synthetic resin mounting
media.
Results:
Argyrophil
granules.…………………….........................
.................................…Dark brown to black
Argentaffin
granules……………………………………
……………………..…Dark brown to black
Nuclei……………………………...............
…………………………………… Red
Background…………………………………
…………………...………………Paleyellow-brown
Figure 3-11 presents the staining of
argyrophil granules by the Grimelius
argyrophil stains.
Fig. 3-11: Grimelius argyrophil staining for
pancreatic endocrine cells. Compare with
the result of staining for the said technique.
Churukian-Schenk Method for
Argyrophil Granules
Purpose: Demonstration of argyrophil
granules in neurosecretory tumors.
Principle: Some processes are argyrophilic
meaning they need a reducing agent in order
to bring the silver to a visible metallic form.
Fixative: 10% Neutral buffered formalin.
Technique: 4 to 5 μm paraffin sections
Control: an argyrophilic-positive carcinoid
tumor is preferred, but a section of small
intestine can be used. Glassware should be
chemically cleaned.
Reagents:
0.3% Citric acid: Citric acid & distilled
water
Acidified water: Distilled water and enough
0.3% Citric acid solution.
0.5% Silver Nitrate: Silver nitrate &
Distilled water
Reducing Solution: Hydroquinone, Sodium
sulfite &
Distilled water (Prepare just before use)
Nuclear Fast Red: Nuclear Fast Red,
Distilled water & Thymol
Procedure:
1. Deparaffinize slides through xylene and
alcohol's to acidified water, pH 4.0 to 4.2.
2. Place slides in 0.5% silver nitrate solution
in a 43 degrees water bath for 2 minutes.
Transfer to a 58 degrees water bath for 2
hours.
3. Rinse slides in three changes of distilled
water.
4. Transfer slides to reducing solution
already heated in the 58 degrees water bath
for 30 to 60 minutes.
5. Rinse sections in three changes of
distilled water.
6. Return sections to the same silver nitrate
of step 2 at 58 degrees for 10 minutes.
7. Rinse slides in three changes of distilled
water.
8. Place slides in the same reducing solution
of step 4 at 58 degrees for 5 minutes.
9. Rinse slides in three changes of distilled
water.
605
10. Counterstain slides in Nuclear Fast Red
solution for 3 minute.
11. Rinse slides in three changes of distilled
water.
12. Dehydrate sections in two changes of
95% alcohol and two changes of absolute
alcohol.
13. Clear slides in three changes of xylene
and mount in a synthetic resin mounting
media.
Results:
Argyrophil
granules.……….……………………….…
…………....................Black
Argentaffin
granules……………………………………
………………………..…Black
Nuclei……………………………………….
..............…………………………… Red
Background…………………………………
.……………………...………………Yellow
-brown
Figure 3-12 presents staining of tissue with
Churukian-Schenk technique for argyrophil
cells.
Fig. 3-12: indicates churukian-Schenk
argyrophil staining of tissue: Compare the
results of the technique with the method’s
coloration.
Bile
Stain
reaction
(Hall)/Fouchet
Purpose: Demonstration of bilirubin in
tissue.
Principle: Bilirubin is oxidized to biliverdin
in an acid medium. This oxidation reaction
is rapidly accomplished by ferric chloride in
trichloroacetic acid medium.
Fixative: 10% Neutral buffered formalin
Technique: 4 to 5 μm paraffin sections
Control: a section containing bile must be
used as a control.
Reagents:
10% Ferric chloride: Ferric chloride &
Distilled water
Fouchet’s Reagent: Trichloroacetic acid,
10% Ferric chloride & distilled water.
Van Gieson’s solution: 1% acid fuchsin &
Saturated Picric acid.
Procedure:
1. Deparaffinize and hydrate slides to
distilled water.
2. Wash sections well in distilled water.
3. Stain sections in freshly prepared
Fouchet’s solution for 5 minutes.
4. Wash slides in tap water, then rinse in
distilled water.
5. Stain sections in Van Gieson’s solution
for 5 minutes.
6. Place slides directly into 95% alcohol and
rinse well.
7. Clear slides in three changes of xylene
and mount in a synthetic resin mounting
media.
Results:
Bile
or
Bilirubin……………………………………
………...…….…Emerald green to olive
drab
606
Background…………………………………
………...………………Yellow
Figure 3-13 shows the Von Kossa staining
of tissue.
Fig. 3-13: shows tissue staining with Von
Kossa’s staining method for tissue section
staining.
1. Deparaffinize and hydrate slides to 50%
alcohol.
2. Rinse slides rapidly in distilled water.
3. Place sections in alizarin red S staining
solution. Check the reaction microscopically
and remove slides when an orange-red lake
forms (30 seconds to 5 minutes).
4. Shake off excess dye and carefully blot
the sections.
5. Dehydrate in acetone (10 to 20 seconds)
and in acetone-xylene mixture (10 to 20
seconds).
6. Clear slides in three changes of xylene
and mount in a synthetic resin mounting
media.
Results:
Calcium
deposits……………………………….……
…………………....…….…Orange-red
Figure 3-14 shows the alizarin red S
staining of calcium in tissues.
Alizarin Red S Calcium Stain
Purpose: To identify the presence of
calcium in tissue.
Principle: Alizarin red S will react with the
cations calcium, magnesium, manganese,
barium, and strontium, but normally only
calcium is present in tissue in sufficient
quantities for demonstration.
Calcium forms an alizarin red s-calcium
complex in a chelation process.
Fixative: Alcoholic fixatives or 10%
Neutral buffered formalin.
Technique: 4 to 5 μm paraffin sections
Control: a section containing calcium must
be used as a control.
Reagents:
Alizarin Red S Staining solution:
Alizarin Red S & Distilled water; Mix the
solution and pH to 4.1 to 4.3 with 0.5%
ammonium hydroxide. The pH is critical.
Procedure:
Fig. 3-14: alizarin red S method for calcium
deposits (orange red deposits).
Rhodanime Method for Copper
607
Purpose: To detect copper in tissue,
especially in liver in Wilson’s disease.
Principle: This procedure is more sensitive
than the rubeanic acid method; however, it
has suggested that Rhodanine demonstrates
the protein to which the copper binds rather
than the copper itself. Therefore, although
considered more sensitive, it may be less
specific than the rubeanic acid methods and
false-positive results may be obtained.
Fixative: 10% Neutral buffered formalin.
Technique: 4 to 5 μm paraffin sections
Control: a section containing copper must be
used as a control.
Reagents:
Saturated Rhodanine solution: Rhodanine &
absolute alcohol
Working Rhodanine solution: Saturated
Rhodanine solution & distilled water. Filter
before use.
Diluted Mayer’s Hematoxylin; Mayer’s
hematoxylin & distilled water.
0.5% Sodium borate: Sodium borate &
distilled water.
Procedure:
1. Deparaffinize and hydrate slides to
distilled water.
2. Using plastic coplin jars, place slides in
50 mL of working Rhodanine solution.
Leave 18 hours at 37 degrees.
3. Rinse slides well in distilled water.
4. Stain slides in Mayer’s hematoxylin for
10 minutes.
5. Rinse slides in distilled water.
6. Rinse slides quickly in 0.5% sodium
borate solution.
7. Clear slides in three changes of xylene
and mount in a synthetic resin mounting
media.
Results:
Copper………………………….…………
……………………....…….…Bright red to
red yellow
Nuclei………………………..……………
……………………………….Light blue
Figure 3-15 represents a tissue staining of
rhodamine for copper deposits.
Fig. 3-15: indicates hepatolenticular
degeneration (Wilson’s disease) of liver
stained with Rhodamine techniques for
copper deposition as seen here with bright
red deposits. These are the accumulation of
copper in the aforesaid disease/tissues.
Bone processing and staining
(Schenk et al)
Bone slabs come in two forms, 1) cortical
type bone and the other, 2) is the compact
bone. These are very strong bones such as
femur (i.e for the cortical bones) or exterior
surface of the flat bones such as ribs, skulls,
trabecular bones, cancellous bones,
epiphysis, marrow cavities, vertebrae and or
the center of the flat bones (for the compact
bones). These are used in bone processing
and bone tissues studies received in the
histology lab and must be processed
accordingly.
Physiologically
and
hiostologically
remodeling of bone takes place more
frequently in young but much less frequently
in old ages: this is a process of constant
dying and repairing of the osseous tissue
608
may be a facilitated process as in pathology
or a normal trend. The remodeling of the
osseous tissue is followed by restoration and
deposition of minerals so that this process
manufactures the bone matrix. Bone consists
of elements that needed for its development
such as cells and organic extracellular
matrix. Failure of these destruction and
repair/remodeling processes result into
disease states that damages bone structural
remodeling such as osteoporosis or
osteodystrophy. Available percentages of
these cells to minerals consist of 70%
minerals to 30-33% organic matters/cells.
An important part of bone is collagen fibers
which is distinctly different from other
tissues collagens. Bone collagen is
mineralized and banded as in form of
lamellae. Respectively cement lines the
proteoglycan ground substance of the bone
where it outlines the fibers. Thus lamellae
(contains concentric layers of bone
surrounding
the
Haversian
system)
demonstrate
the
micro-anatomic
structure/pattern of the bone and other
structures such as Haversian canals,
canaliculi and lacunae (contains osteocytes)
in compact bones, Recalling from our
histology and anatomy for the purpose of the
bone tissue structural visualization: we have
the Haversian and non-Haversian systems
each consists of lamellae roughly parallel to
the surface of the bone and concentrate
channels of Volkmann’s canals contains
one or more blood vessels respectively;
figure 3-16 shows a Haversian system in
osseous tissue This figure shows a system of
osteon, Volkmann’s canals, and bone cells
in lamellae in cross section.
Fig. 3-16: a photomicrograph of Haversian
system in osseous tissue in compact bone, a
system of osteon, canaliculi, lacunae,
lamellae and black spaces containing
osteocytes. Note the haversian/canal/system
of osteonic tissue in cross sections.
Magnification 100x decalcified bone.
Histologically there are three different kinds
of bone cells: 1) osteoblast responsible for
making bone, 2) osteoclasts for bone
destruction, and 3) osteocytes which are
the bone maintaining cells. These cells are
responsible for bone remodeling and their
interplay result into remodeling of bone. The
osteocytes reside in a place called
“lacunae” within the bone. Still with
regards to these cells, we have ACP (Acid
Phosphatase enzyme), which is found in
the osteoblasts, and ALP (Alkaline
Phosphatase) found exclusively in
osteoclasts
(refer
to
clinical
chemistry/enzymes part one) detectable in
bone with aids from histochemistry enzyme
studies we demonstrate tumors associated
with the ACP and ALP enzymes and their
cell origin.
In this respect bone minerals include
calcium (as in calcification), phosphate
609
where it admixes or combines with hydroxyl
ions to form hydroxyl apatite, which is the
chief
structural
element
of
bone.
Additionally, due to presence of 38%
minerals such as calcium in bone,
amorphous phosphate would form. If we
add hydroxyl ions to amorphous phosphates
there will be formation of hydroxyl apatite.
This may be a form of crystal lattice
formation (spherulite) in which citrate,
carbonates and fluoride ion (as others such
as magnesium and potassium, including
other minerals etc.) may be substituted or
included as for phosphates.
Ossification (bone formation) is the process
of bone formation and development is
divided
into
the
following
two
classifications:
1. Intramembranous ossification
(i.e. skull, sternum and pelvic bone)
and, this occurs in flat bones, its
mesenchyme cells transforms into
osteoblasts with subsequent bone
formation.
2. Endochondral ossification (i.e.
long bones and major parts of
skeleton). This occurs in most long
bones of the skeleton and the
mesenchyme cells differentiate in
this type of ossification. This
develops and lay down in cartilage
forms and then develops into final
shape of the bone itself.
Additionally, with tissue processing
techniques, bone technique is more difficult
than other tissue procedures (due to hardness
of bone and its processing) and should be
attended with care. With regard to bone
techniques: for the decalcified bone, frozen
sections, paraffin and celloidin sections
must be used. For mineralized bone
(containing minerals), frozen section, plastic
tissue blocks must be used for microtomy or
saw/ground/grinding section. In this
connection mineralized bone sections can be
used
for
microradiographic and
histomorphometric studies.
Bone biopsies are applications that use for
identification of metabolic bone diseases
(MBD),
tumors
(osteosarcoma),
hematopoietic
diseases,
infections,
osteomalacia/rickets,
osteoprosis
and
osteodystrophy. Bone slabs and
amputations routinely uses for tumors,
gangrene, chronic osteomyelitis and
osteochondritis. Respectively, resection
specimens are mostly used for benign or
low-grade malignant osteoid. And
finally mineralization is determined for the
purpose to ascertain the type of treatment is
available to offset these above changes.
Bone processing
(Schenk et al &
Bancroft et al)
For bone processing decalcification of tissue
slabs must be done, this must occurs before
tissue cutting and microtomy or after
tissue fixation. Most of time, this is done
subsequent to fixation. This can be
accomplished by a mild or strong acid such
as formic acid, nitric acid or hydrochloric
acid, etc. Before this process began tissue
have to be fixed by 10% NBF (Neutral
Buffer Formalin). NBF fixative is suitable
for MMA (metamethyl acrylic waxes)
sections as well. Some fixatives such as
Zenker’s solution, Carnoy’s fluid or B-5 are
toxic to the tissue and may damage it.
Therefore must not be used.
In regards to sawing of the bone slabs in
preparation to subsequent processing stages,
this can be processed by selecting a type of
or handyman’s bench band saw, etc:
with this the first cut is done through the
610
mid-plane of the bone and then this can be
proceeded by cutting parallel to the first cut:
a 3-5 mm thick slabs are cut and prepared.
The tendons and soft tissues attached to the
bone taken from the diseased patient must
be removed before sawing/cutting the slabs
and can be fixed additionally to 24-48 hours
of fixation. A saw guide plate or a wood
block held against the first cut edge insures
an even slice. After fixation we decalcify
bone by immersing it into a weak/or strong
acid as cited above. The purpose of
decalcification is to remove calcium from
the tissue and leave it with a minimum
artifacts and an ease on microtomy. Calcium
(The American heritage science dictionary)
is found in teeth, bone and other calcified
tissues such as T.B. (tubercle, bacilli
affected tissues), atheroma or atherous
tissues (as in atheromatosis), and in tissue
infarction
such
as
Gandygamn
phenomenon and cardio vascular
calcification (Miller et al).
The 33% of the osseous tissue is composed
of cells, fibers and ground substance as
collagen (vide supra) and the rest are other
elements occupying the bone matrix as
calcium, phosphate or etc. Decalcification
may be done by formic acid, nitric acid
or hydrochloric acid. Decalcifying fluids are
employed for such purpose and is composed
of different solutions. This decalcifying fluid
are named as Godding’s Stewart’s fluid
with formic acid content and formalin, or
van Ebner with hydrochloric acid and or
EDTA (chelating substance) known as
“versenen or squestrene”. It will take a
long time to decalcify however EDTA can
result in an excellent decalcification. The
ratio of tissue to decalcifying agent must be
maintained at 20-50 times (X) of the size of
the specimen.
An optimal decalcifying solution must have
the following properties:
1. Act quick on the bone
2. Not interfere with subsequent
process of staining procedures, etc.
3. Must not damage tissue, it
should be as little damaging as to the
tissue(s) as possible,
4. As thoroughly remove the calcium,
To test bone decalcification is important
and it is crucial to know the end-point of
decalcification as well. This may be done by
the following procedures:
1. Radiographic x-ray of the tissue
(excellent)
2. Chemical end-points such as
saturated lithium carbonate solution
or 5-10% sodium bicarbonate
solution for several hours or sodiumoxalate solution
3. Probing
and
needling
the
decalcified tissue (the worst)
As with chemical end-point following
decalcification, tissue may be rinsed with
running tap water, this may take for small
tissue sections for 30 min to 1-4 hours for
large pieces. The most popular chemical
end-point testing for decalcification in
Canada and or North America is sodiumoxalate: in this technique: the end of the
decalcification (end-point) will be
performed when decalcification has reached,
this procedure applies when the end point
has reached and we follow as: to 5.0 ml of
decalcifying fluid, add concentrated
NaOH or NH 4 OH to pH = 8-10 and then
add 1.0 ml of K or Na oxalate: therefore if
there is any cloudiness in the solution it
indicates presence of calcium ion. The
611
following chemical formula (Eq. 1-1)
explains this chemical process:
Eq. 1-1:
O 4 + 2Na
Ca   + Na 2 C 2 O 4 → CaC 2
By addition of alkali there may be
cloudiness, indication of presence of
calcium ion: if the solution is clear it
indicates complete decalcification. Tissue
photoradiograph is an excellent form of
end-point testing however it is expensive
and is radiopaque with tissue treated with
mercury fixatives. There are chemical
decalcifying agents available commercially
and are known as RDO®, Cal-Ex® and
decalcifying fluids etc. Physical testing of
end-point examination such as probing and
needling are not recommended and are
damaging to the tissue section.
Attention: Bone fixation for metabolic
bone disease studies need to be fixed
with formaldehyde fixatives using Millonig
buffer (sucrose, NaOH, NaHPO 4 or NaH 2
PO 4 /sodium monophosphate or sodium diphosphate) and un-decalcified bone is
assayed.
Employ
agitation
for
decalcification process: this included in
factors that affects a better decalcification
which are the temperature (best is at room
temperature), agitation, and complete
suspension of bone into the decalcifying
fluid to increase the rate of decalcification.
Still with bone processing we have,
subsequent to fixation of the bone sections
and decalcification we do dehydrate the
bone tissue sections with the aforesaid
dehydrating fluids/agents (such as grades of
alcohols). Next after this we have clearing of
the tissues with clearing agent mentioned
above (such as xylene and acetone or xylene
substitutes) in here the best suited for the
situation is xylene or acetone or xylene
substitutes. This step leads to microtomy
where paraffin with added plastic resins
polymers (i.e. iliac crest trefine biopsies)
may use paraffin sections and embedding
whereas undecalcified bone slabs may be
embedded with/in MMA/Methamethyl
Acrylate, when MMA has been added with
ethylene glycol it is known as GMM (Glycol
Methamethyl Acrylate), the undecalcified
bone procedure are for the purpose of
studying bone with the its minerals
components intact. Therefore there is no
need
of
decalcification
procedures
mentioned for the decalcified bones: the
knife or blade is special in these types of
sectioning and cutting, we used tungsten
carbide tipped blades/knife for undedalcified
bone. For processing of the undecalcified
bone tissues we can utilize acrylic resins or
plastics as MMA or GMM (kernacs et al).
These are firmer in consistency and hold the
hard tissues reasonably well without any
breaking down of the paraffin wax or other
conventional waxes used for other tissue
processing.
In here the general principle of microtomy
as the next stage in the process of the bone
tissue sectioning and ribbon sectioning will
be covered. This part of microtomy is
similar
as
other
tissues
cuttings,
nevertheless, due to nature of this tissue
obviously proper care must be applied:
longitudinal sections of the cortical bone
may section better when bone is oriented at
right angle to the blade/knife and “cut
along the grain” for this type of tissue a
sharp blade is mandatory and is the key to
easy
ribbon
sectioning,
following
microtomy, tissue ribbon sections must be
selected and floated into the water-bath with
flattening and adhesion: for mounting,
resinous mountant (mounting media) such
612
as resin polymers, or poly-lysine coated
slides are popular.
The following terminal concept of bone
tissue section would be hydrating and
dehydrating for the subsequent stages and
finally staining of the tissue sections, a
couple of staining techniques are included in
here (most common methods): this embraces
the decalcified bones and mineralized
bones (undecalcified).
The compilation names of staining methods
for decalcified bones compasses H & E
staining, staining for collagen and staining
for mucopolysaccharide as mentioned under
mucosubstances, these stained include PAS
and reticulin staining methods, the above
decalcified staining methods can be
processed and stained as mentioned for other
tissue processing sections. Figure 3-.17 is H
& E staining of the decalcified bone
section viewed under photomicrograph.
Also it follows the most common staining
bone
Fig. 3-17: A 5 micron thick section of H &
E staining of mature bone. Specimen was
decalcified in a 5% formic acid prior to
paraffin embedding. The hematoxylin
section stained with Gill’s II hematoxylin
and counterstained with Eosin Y/phloxine
stain (H & E). This is a popular staining
method
in
histopathology
and
histotechnology lab.
Tissue
sections
with
undecalcified
mineralized bone sections: these include
such staining methods as frozen section
staining with H & E stains and for MMA
sections (for mineralized sections): we have
the availability of H & E, solo-chrome
cyanine method and modified Von Kossa
staining method (mentioned below/vide
infra) for undecalcified bones. Following
the undecalcified stains citation, I will
mention the mineralized bone staining
procedure as in modified Von Kossa
methods, these are described subsequently.
Figure 3-18 shows the staining of
undecalcified bone sections with the Von
Kossa staining method (Villanueva et al).
Fig. 3-18: Von Kossa staining of
undecalcified mineralized bone sections
with
MMA/GMM
embedding
with
demonstration of black field showing
mineralization areas of the healing bone
with two micron thickness.
613
Modified Von Kossa's methods
(for calcium deposits modified for plasticembedded sections of bone/un-decalcified
bone)
Fixation: 70% alcohol, 10% buffered
formalin.
Solutions
Aqueous silver nitrate, 5%
Aqueous sodium thiosulfate, 5%
Mayer's hematoxylin
Basic fuchsin in 70% ethanol, O.l%
Procedure
l. Deplasticize in xylene for 4 hours at 55°
C.
2. Transfer to
100% ethanol
100% ethanol
95% ethanol
95% ethanol
75% ethanol
Distilled water
3. Incubate in silver nitrate solution at room
temperature under ultraviolet light for a
minimum of 6 hours.
4. Wash thoroughly in distilled water for 3
to 5 minutes.
5. Rinse in the sodium thiosulfate solution
for 2 to 3 minutes.
6. Wash thoroughly in distilled warer for 3
to 5 minutes.
7. Stain in Mayer's hematoxylin for 15
minutes.
8. Wash in distilled water for 5 minutes.
9. Transfer to 70% alcohol for 1 minute.
10. Stain in 0.l% basic fuchsin for 15 to 30
seconds.
11. Dehydrate and clear in the following:
95% alcohol
l0 dips
100% alcohol
15 dips
L00% alcohol
15 dips
Xylene
20 dips
Xylene
20 dips
Xylene
1 min.
12. Mount in Eukitt's mounting medium.
Result
Calcium deposits-dark brown to black
Osteoid seams-bright pink
Nuclei-purple
Cytoplasm-pink
Fluorescein labeling also is used in bone
tissue screening and staining, the
antibiotics (tetracycline/with fluorescent
property) may be labeled with calcium to
fluoresce in the tissue section as the
principle tenet of the fluorescein staining
method.
A short description of morphometry of
bone will be mentioned in here; this is as
one of the special techniques for assessing
the bone minerals, it consists of quantitative
and qualitative assessment. As a major
advantage of this method is to study and
evaluate Metabolic Bone Diseases
(MBDs)
such
as
post-menopausal
osteoporosis,
osteomalacia,
osteitis
deformans and osteodystrophy. The
fluorescent techniques mentioned previously
involves in these evaluation, once the
fluorescent dye in form of tetracycline
antibiotic injected into subject/patient with
MBDs prior to sampling (iliac crest biopsy)
at a certain time, the fluorescent labeled
conjugate will be found in the bone tissue
(after the iliac or sternal biopsies): this
fluorescent antibiotic parts with the calcium
and other minerals and fluoresces in the
sample: therefore a subtle study may be
performed to measure the minerals. This is
called “bone histomorphometric analysis.”
During histomorphometric analysis, an
assessment of relative amount of
trabeculae, osteoid, restoration and
deposition (apposition) can be made through
photomicrgraphic observation and
614
analysis. These studies may be done with
manual, or semi-automated or fully
automated instruments. With this method we
can relatively estimate the degree of
remodeling due to changes in osteoblasts,
osteocytes and osteoclasts and minerals:
cells and elements responsible for
remodeling of bone tissues.
Amyloid substance staining
Amyloid substance is a derivation from
some proteins and carbohydrate in partly of
immunoglobulin nature. The carbohydrate
nature consists of polysaccharide and
mucoploysaccharides contents of 1-5 %. The
substance historical discovery was marked
by the work of Virchow (1853)
demonstration of the amyloid materials
known to that day by name “waxydegeneration”. Today it is quite recognized
that there are different types or classes of
amyloid. The classification of amyloid
substance still not straight forward and is
haphazard, but although exists. Amyloid
substance such as “amyloid-P and
amyloid-A” (P derived from plasma and
serum amyloid A) or “amyloid-S” (derived
from serum) are widely recognized along
with other numerous amyloid substances
indicated by their location of affection or
origin. Amyloids are fibrous proteins and
carbohydrate with molecular structure
forming β-pleated sheets. The following
criteria apply to detection of amyloid
materials (Bancroft et al & Kiernan et al):
1. These are amorphous materials
that are predominantly extracellular,
eosinophilic substance
2. Upon detection with Congo red stain,
it gives an apple green positive
birefringence (faintly refractile)
matter with emission action of
polarizing light
3. It has a beta-pleated sheet structure,
and
4. Has a fibrillary ultrastructure
pattern.
Amyloid substance traditionally used to
cover and deposits in organs and tissue after
their long term formation in disease states as
in syphilis and T.B patient in the past
nevertheless in twentieth first century its
transformation commonly occurs in diseases
such as chronic inflammatory conditions:
i.e. tuberculosis, rheumatic arthritis (RA),
rheumatic fever and heart disease
(RF/RHD), diabetes and in most recent era
the
debilitating
illness
such
as
Alzheimer’s disease. The amyloid term
was originally coined to signify the reaction
of amyloid substance with iodine dye
reaction which ultimately used to yield a
“violet color” with carbohydrate and
mucopolysaccharide, thus the term
“amyloid” was named for such materials
and reaction with amyloid.
Amyloid substance due to its long duration
of occurrence is quite difficult to detect and
identifies during the affected person’s life
span/time nevertheless tissue biopsies from
the affected organs which are safely biopsyable (such as kidney, liver, spleen or etc.)
may provide a mean to diagnosis the
condition, although organs such as brain and
heart are quite difficult to be biopsied and
therefore escape the detection option.
Therefore a solid detection cannot be
established in such cases for inaccessibility
to biopsy them.
Recently a heavy
accumulation of such materials in certain
organs such as rectum (rectal mucosa) can
be obtained through rectal mucosal biopsies
or so on. The fact is that if the test
615
(histochemical staining) is negative this
will not preclude the presence of the
illness/amyloid substance due to error in
sampling as it always exists. This substance
has a unique fibrillary structure aside
from other fibers, which is independent from
the original anatomic site.
For classifying amyloid substance: a current
classification
includes
1)
primary
amyloidosis or amyloids, this is a
localized deposition in some mesodermal
tissues origins such as kidneys, heart, liver
and skin. And 2) secondary amyloidosis
or amyloids affecting a preexisting
pathological conditions such as chronic
inflammatory
conditions,
diabetes,
rheumatism (as in RF, RHD and RA) or in
recent era with Alzheimer’s disease, which
may lead to a systemic involvement and
deposition. In Alzheimer’s disease it may
manifests to organ involvement and
neurofibrillary tangles (this is covered in
neuropathology
and
neurohistotechnology at the next chapter
(4) in this part five/vide infra) and may form
in addition to yielding of dementia and
amentia, with ultimate organ failure, with
many years prior to the commencing of
acute and clinical apparent disease.
Pathophysiology of amyloidosis include
self-aggregation and polymerization of the
amyloid proteins due to the presence of
large proportion of beta-pleated sheets
secondary to conformation after the
precipitation events, this may be evident in
such disease as Huntington disease, α 1 antitrypsin deficiency, the plasma Serpin
disease, prion diseases, and certain
hemoglobin
and
neurofibrillary
degenerative diseases (Bancroft et al.).
Diagnosis depends upon biopsies of the
biopsy-able organs and processing and
staining of tissues obtained in such a way by
special
histochemisrty stains such
Congo red and Sirius red stains.
as
As said it is an amorphous, extracellular,
eosinophilic, faintly refractile substance or
aggregates in the affected tissues or organs.
The following figure 3-19 shows a closely
taken (close-up) photomicrograph picture of
an amyloid substance in specific tissue
section as in bone (Highman’s Congo
red is specific histochemical stain for
amyloids). Sirius red technique may be used
as well to stain this substance. A control
slide must run with the test slides so as to
avoid the fainting of character of the
substance after primary stain preparation. As
for immunohistochemistry the use of
immunoperoxidase/DAB
(Diaminobenzedine
Hydrochloric
Acid/which is carcinogenic) method may be
applied for such staining study purposes in
tissue sections.
Fig. 3-19: (a) is the photomicrograph of
amyloid substance stained with Congo red
and (b) is the cross polarization of tissue
with polarizing microscope: note the apple
616
green, faintly refractile (birefringence) of the
amyloid fibrils substance present in the
sections
of
decalcified
bone
(spherulites/liquid crystalline structure).
Chapter Four (4)
Histopathology techniques in
neurology (staining properties)
(Kiernan et al and Bancroft et al)
I
n the past due to numerous histologic
staining
methods
used
in
histopathology and histotechnology in
science of neurology confusion existed,
however today several more reliable
techniques have been introduced in
histotechniques to overcome the drawbacks
of conventional histochemical staining
methods. These new generations of tests are
known today as immunohistochemical
staining methods. Diseases such as
dementia,
amentia,
and
neurodegenerative diseases routinely
are investigated in pathology laboratory to
diagnose these pathologic entities therefore I
will explore these abnormalities with a short
description. The approach will be a general
definition of tissue processing, staining and
petite elaboration of neuropathological
states as would be a part of the histology
texts.
regard silver impregnation
techniques in neuropathology is a cliché
method
in
comparison
to
immunohistochemistry nonetheless is still
considered an elaborate and invaluable
procedural tool in histotechnology of
nervous system and used in many labs.
In
this
Gold toning with gold chloride added in
some times in the past to silver nitrate
staining methods still is a common
techniques employed in this method for
nervous system staining (Formmann -1864
and Ranvier 1868). Metal impregnation
however is a strong tool in the hand of
histotechnologist
for
microanatomical
investigation into neurological pathology. A
short description of the nervous system as
far as it is needed to impart these ideas to
histotechnology will be covered and will be
reviewed and warranted in here;
Nervous system mainly composed of
neurons, which is the single cellular unit of
nervous system, neurons are divided into
3 main aspects, 1) is the body cells, 2)
dendrite, and 3) axons. As will be briefly
elaborated, these are constituents of the
nervous system. The cell body is the
cellular dimension of neurons as it is
composed of cytoplasm, nucleus, pigments,
inclusion bodies and Nissl granules. Nissl
granules are in fact the RNAs resides on
the rER (rough endoplasmic reticulum) in
the cytoplasm of the neurons. Due to its acid
nature RNA would be studied with basic
dyes and demand to be stained by basophilic
stains such as basic dyes, including the
followings: neutral red; cresyl-fast-violet;
toluidine; thionin; pyronin and methylene
blue (for vital staining).
The following terms applies to cell body and
its contents at normal and abnormal states:
chromatolysis apply to the dislocation of
Nissl granules from its normal nuclear
periphery to surrounding cytoplasmic
periphery,
which
is
an
abnormal
phenomenon, other inclusions such as
pigments: i.e. neuromelanine
and
lipofuchsin are found in the cell body along
617
with substantia nigra and lipofuchsin can be
found in old age individuals respectively.
With spanning to dendrites and axons,
dendrites are ramified extension or
processes of the cell body as divisions so as
to receive electric signals from the axon of
another neuron(s) passing the signals
through cell body’s fibers toward cell body
axon. The junction between axon with other
axons or dendrites are connected by means
of a space called” synaptic cleft”, where the
electric signals transforms to chemical
signals by means of neurotransmitters.
Along the axon-fiber exists the Schwann’s
cells and myelin sheet with connecting node
called “node of Ranvier”. The axons and
dendrites (neurofibriles) are coated with
myelin sheet and Schwann’s cells, which
breaks at the node of Ranvier. In a
neurodegenerative
disease
such
as
Alzheimer neurofibrillary plaques and
tangles forms and it occupies and affects all
internal cell body fibers along with external
fibers with deposition of amyloid substance.
After
this
brief
anatomic
and
pathophysiology of the nervous system
comes tissue processing and staining
techniques for Nissl granules and
neurofibrillary materials. These include
techniques as H & E method with routine
tissue processing (vide supra). This may be
Van Gieson staining method which are
popular and demonstrate a crispy
cytoplasm. Vascular changes as well as
advantages of staining the myelin sheet are
satisfactory.
Quality of staining and detection of Nissl
granules staining techniques as mentioned
above as with other staining techniques
depends on factors such as temperature of
the reaction, PH of the surrounding, the
duration or length of differentiation in
regressive staining of these tissues. As with
staining the anterior horn of the spinal
cord as one of the main part of the CNS, it
may stain by the above (stains for Nissl)
stains. In addition the nervous tissue fixed
with alcohol may be stained particularly
well with thionin stain. Figure 4-1,
indicates anterior horn of spinal cord
staining (vide supra). This structure is rich in
Nissl granules.
Fig. 4-1: Anterior horn of spinal cord with
silver staining: note the dorsal root
gangilion.
The following Creysl-fast- violet stain
procedures and method is for paraffin
sections:
Staining of Nissl granules with
cresyl-fast violet:
Procedure
Nissl substance staining
Nissl granules are distributed in the
cytoplasm and dendrites of neurons.
Nissl granules can be demonstrated with
many basic dyes:
Neutral Red
Methylene Blue
618
Toludine Blue
Cresyl Fast Violet
Staining with Cresyl Fast Violet
Depending on the pH level of the cresyl
violet stain and the extent of
differentiation, this stain is able to highlight
Nissl substance only or include the nuclei of
neurons and glia.
Method for Fixed/paraffin embedded tissue
Cresyl Violet Solution
Cresyl Fast Violet 0.025gm
Distilled water 100ml
pH to 3.8 with Glacial Acetic Acid
If using cryostat sections, fixed in 10%
Neutral Buffered Formalin for 5 minutes
before staining. There is no need to dewax!
Dewax slides and bring to water
Place sections into cresyl violet acetate
solution pH3.8 at 37ºC-10 minutes
Differentiate in 95% ethanol
Dehydrate, clear and mount in DPX
Note: Cresyl Violet Acetate and Cresyl Fast
Violet ARE THE SAME.
Results: Nissl Granules-Purple
In
immunohistochemistry cellular
markers in tissues can be utilized for
immunohistochemical stains, these can
differentiate neuroendocrine tissues on
the basis of immune-reactivity with the
corresponding antibodies. Some of these
markers include: first the normal
cytoskeletal proteins such as NF 70 (L),
and or NF150 (L), etc. these markers are
filaments specifically to nerve cells with
their corresponding antiobodies. The
second group of markers is named
cytoplasmic specific protein for nerve cells
such as PGP9.5 and the neuron specific
endolase (NSE), this marker is not solely
specific for nervous tissue. The third groups
of markers are neurosecretory granules
associated proteins, (i.e. as chromogranin
and synaptophysin), these are associate with
neurosecretory granules and synaptic
membranous glycoprotein respectively.
Antibodies to these markers are well
distinguished and are available for
immnuohiostochemstry
by
several
manufacturers. In regard to axons and
dendrites there are some specific
immunohistochemcial staining methods as
Bielschowsky’s silver nitrate staining
for the neuronal processes. This method uses
ammoniacal silver nitrate solution (vide
supra). The Palmgren’s method is
primarily used to staining the axons of the
peripheral nerves (palmgren-1948).
As with degenerative nerve fibers, we have
formalin fixed specimen with uranyl
nitrate before impregnation with silver
nitrate in an alcoholic solution treatment:
this is for detection of plaques and tangles in
Huntington’s disease and Alzheimer’s
disease. The method use this treatment
includes silver nitrate staining as in Eager’s
staining technique. Degenerative axons
also may be detected by teased fibers: these
fibers may appear in cluster of gray to black
globules. Nerve fibers may also stain by
vital staining with methylene blue as similar
to other tissue vital staining. With addition
to myelin staining, the myelin is formed
from oligodendrocytes and Schwann’s
cells in CNS (central nervous system).
Myelin is made up of cerebrosides and
lipids. A good example chemical staining
method for myelin staining may be Weil’s
method for myelin staining as a candidate.
This is a hematoxylin technique at the
end.
Still with oligodendrocytes (of nervous
system) as mentioned earlier are located in
the white matter of the brain and are
myelinated, for this we can make use of the
619
staining properties of Penfield’s combined
method for oligodendrocytes and microglial
cells as well.
With other parts of nervous system such as
staining for microglial cells, ganglial cells
and astrocytes (majority are located in the
cerebral cortex/gray matter) which are the
supporting cells of the CNS (Central
Nervous Sysytem), we have Cajal’s gold
impregnation and staining (as in Cajal’s
gold sublimate) method. These cells are
important in neuropathology for their
malignant and benign tumor forming
capacity. Another stain for supporting cells
is PTAH (Phosphotangstic
Acid
Hematoxylin stain) for astrocytes
recovery in the tissue sections. Figure 4-2
presents astrocytes stained with PTAH.
Fig. 4-2: Cajal’s gold impregnation and
staining of astrocytes as stained black, more
details in the text (vide supra)
The following is the
procedure for astrocytes:
Cajal
staining
Astrocytes: Cajal Stain
Purpose of the stain: The demonstration of
astrocytes. This method has been replaced to
a great extent by immunohistochemical
procedures.
Principle of the stain: Astrocytes are
selectively stained with the Cajal gold
sublimate method on frozen sections.
Fixatives: Formalin ammonium bromide for
no less than 2 days and no more than 25
days. If the tissue has been fixed originally
in 10% Neutral Buffered Formalin,
wash and place in formalin ammonium
bromide for 48 hours before proceeding with
the technique.
Sectioning: Cut frozen sections at 20-30
microns. Do not pick up on slides; the
section should be free-floating for this
technique. Tissue will section better if
washed in tap water for 30 minutes before
freezing.
Controls: Use a section of cerebral cortex
(not spinal cord) for the demonstration of
astrocytes.
Reagents:
1.
Formalin
Ammonium
Bromide:
Ammonium bromide, Formaldehyde 3740% & Distilled water.
2. Gold Sublimate: 1% Gold Chloride, 1%
Mercuric chloride & Distilled water.
3. 5% Sodium Thiosulfate (Hypo): Sodium
thiosulfate & Distilled water.
Procedure:
1. Wash the free-floating frozen section
in several changes of distilled water.
2. Transfer the sections to gold sublimate
solution, and leave in the dark for 4 hours.
The sections should be purple.
3. Wash well in distilled water.
4. Treat with 5% Sodium thiosulfate for
2 minutes.
5. Wash section well in several changes of
distilled water.
6. Carefully mount the sections on slides,
blot with bibulous paper, and dehydrate in
95% & 100% alcohols.
620
7. Clear in xylene and mount in synthetic
resin.
consult one of the references cited at the end
of this section.
Results:
Astrocytes
with
perivascular
feet….………………………………………
…………..Black
.
Looking into dementia and amentia: they
occur mostly in neuronal degenerative
diseases as Huntington’s disease or
Alzheimer’s. Dementia is classified into
three types: 1) vascular dementia due to
cerebral infarcts, 2) dementia due to
presence of Lewy bodies or known as
“cortical Lewy bodies”, this happens in
Alzheimer’s and possess cerebral-cortexinclusion-bodies, the cortical Lewy bodies is
the term applied for this condition, and 3)
dementia of frontal and temporal lobes
with neural loss in these anatomic areas. For
Alzheimer’s silver nitrate impregnation as in
Bielschowsky technique is superior although
underestimates amyloid in sections.
Immunohistochemistry may help to
obtain and catch up with these histochemical
staining
shortcomings.
Neurofibrillary
tangles and plaques may be demonstrated
with application of thioflavin-S method: in
this we use fluorescent dye(s). Still with
neuropathology
staining,
modified
Bielschowsky has good application. Other
staining methods such as methanamine
silver staining for the detection and
presence of amyloid substance may be used
however detects few tangles, but is not
tangle sensitive.
At this end I suggest for the detail coverage
of the topics in regards to specimen
handling, organ cutting and preparation as
brain, spinal cords and peripheral
nervous system biopsies and processing
Neuroendocrine staining and
related stains
(Kimura et al and Soga et al)
The neuroendocrine system
Neuroendocrine system is a diffused system
of endocrine glands and nervous system.
This system is composed of vesicular
membrane glycoproteins in nature that
facilitate
inter
and
entracellular
communications. This is accomplished
through an organized system of glandular
tissue organs and nerve cells. Most common
organs and tissues involved in the process
and in endocrine system are classified as
skin, brain, large and small intestines
(enterochromaffin cells/EC serotonin stained
with Masson trichrome), pancreas, adrenal
glands, pituitary gland (hypophysis) and
thyroid gland.
Originally nerve fibers signal the electrical
impulses in a space called “synaptic cleft” or
pre-synaptic vesicular granules through
chemicals known as “neurotransmitters”.
The released substances now called
neurotransmitters acts physiologically and
functionally as signal conductors with amine
or peptides in nature, i.e as tryptophan
when
decarboxylated
to
5hydroxytryptomine or commonly known as
“serotonin”. These neurosecretory peptides
may be found in autocrine (exocrine) or
paracrine (endocrine) glands distributed
throughout in the above cited organs and
tissues.
Regulatory
functions
of
neurosecretory
granules
glycoprotein
peptides and amines are to coordinate and
organize bodily functions and tissue
621
communications: this includes classical
neurotransmitters of neuronal tissues.
These tissues and organs are regulated by
means of this collateral and mutual
collaboration between nervous system and
endocrine system. In this regards, the
regulatory peptides and amines released
form the neurosecretory vesicles inner
membrane are distinguished and classified
as the followings: 1) Amines, 2) peptides (as
tryptophan), 3) chromogranin A, 4)
synaptophysin, 5) NSE or NeuronSpecific Endolase, 6) GPG9.5 (or
commonly known as protein gene product
9.5, and lastly 7) intermediate filaments
[these classification has been mentioned in
neuropathology section of this part five
(chapter
4)].
Neuroendocrine
cells
sometimes used to be named as APUD or
Amine Precursor Uptake Decarboxylation
for
they show
L-Dopa
and
5hydroxydecarboxylates and they produce
serotonin and catecholamines.
neurohypophysis secretory granules and its
peptide products such as oxytocin and
vasopressin (ADH/Antidiuretic Hormon)
may be stained by the performic acid-alcian
blue stain or technique. The modified
Gomori-chrome hematoxylin and
aldehyde thionin will stain these glandular
product portions of neurohypophysis as
well. As in the adenohypohysis stains such
as PAS and OFG (orang G acid- fuchsin
green) differentiates acidophilic and
basophilic cells in the pituitary (these are
three types of cells: Chromophobe cells,
acidophilic and basophilic cells, 1)
acidophilic cells are GH and prolactine, 2)
basophilic
cells
are
thyrotrophs,
corticotrophs and gonadotrophs, and 3)
chromophobes are mixtures of the other two
however show no cytoplasmic staining with
a little differences. The stain such as MSB
(Martius Scarlet Blue) will stain both
acidophilic and basophilic cells with no
differentiation. A cocktail of stains such as
performic acid-alcian blue-PAS-OG
The following endocrine organs and tissues
have proceeding histochemical stains (such
as
silver
nitrate
techniques
for
neuroendocrine
cells)
characteristics/reactions. Pituitary gland
consists of pars distalis, pars intermedialis,
and pars tuberalis (or known as anterior,
pituitary, stalk and posterior respectively).
The gland is divided into posterior, middle
and anterior portion, which are respectively
called,
adenohypophysis,
stalk
and
neurohypophysis. With adenohypophysis we
have secretion of peptide hormones such
as, GH, FSH, LH , TSH, ACTH, and
prolactin (for detail refer to clinical
chemistry section part one chapter in organ
systems), Additionally neurohypophysis
contains neuronal fibers originating from the
hypothalamus through pituitary stalk to
these posterior end of the gland. In
(orang G) and carmoisine staining
techniques may differentiate cells to
basophilic
and
acidophilic
class
respectively. In this connection these
staining are significant in detection of
adenomas of the pituitary gland: these
specific stains for somatotroph and
lactotroph cells include acid stains and
carmoisine erythrocin with positive results.
As for corticotroph cells stains with PAS
and bromine-AB (alcian blue) and finally
the thyrothrophs and gonadotrophs cells are
stained variably with PAS: these cited
staining
reactions
embrace
adenohypohysis portion of pituitary
gland.
Also pancreas (Soga et al) is named as a
neuroendocrine organ it consists of
endocrine
(paracrine)
and autocrine
622
(exocrine) cells. The secreting cells of
endocrine cells are located in the “Islet of
Langerhans” originating in B-cells or βcells producing insulin, as where A2cells or α-cells secreting glucagon and Dcells or delta cells or A1 cells/gamma cells
making
the
somatostatin
hormone.
(Remember C- cells or colloidal cells are
located
in
thyroid
parafollicular
cells/colloidal cells/ these are stained with
Sevier-Munger technique: this tissue is
engaged in making iodine for calcium
production in situ). Somatostatin is produced
and secreted by other endocrine organs or
tissues such as brain, hypothalamus and
pituitary. These hormones can be
demonstrated by means of histochemical
stains as silver (Ag) techniques (Grilmelius/
Rudbeck laboratory et al), or aldehyde
fuchsin and or chrome- alum- hematoxylinphloxine. Immunohistochemistry may be
used to detection of pancreatic hormones.
The other organ which is an endocrine gland
is the adrenal glands they contain
neurosecretory cells busy making adrenaline
and noreadrenaline hormones (refer to
clinical chemistry part one chapter on organ
systems). This has been discussed under
neuropathology, etc. (vide supra) and in
short this gland has made of chromaffin
cells that stain with argerophil and
argentaffin techniques. It is used for
identification of tumors of chromaffin
producing cells, these tumors are as; 1)
pheochromocytoma (tumor of adrenal
medulla), and 2) neuroblastoma (highly
malignant tumor), this tumor originates from
primitive neuroblasts.
In this respect cartoid bodies and lung tissue
are also both engaged in endocrine activity.
Both have neurosecretory granular reactions.
Tumors of carotid bodies such as
paraganglioma and for lung, oat cell
carcinoma may be differentiated on the basis
of histochemical reactions and based on
specific stains. Carcinoids (Soga et al) are
tumors of a low grade malignant cancer like
tumors/cancers arising from gut/intestinal
tissues and rarely may derive from lung or
other likely tissues these are benign and
malignant tumors or neoplasms of endocrine
system enterochromaffin cells which rise
to carcinoid tumors was identified first by
Kulchitsky in 1897, although they may be
found in other tissues. The malignant
carcinoids may metastasize to liver which
this may release many amines and peptides
in the systemic blood stream/circulation,
NETs (Neuroendocrine Tumors) are
incorrectly subsumed as carcinoids. This is
called “carcinoid syndrome” with certain
specific signs and symptoms. Figure below
(Figure 4-3 shows neuroendocrine tumor
(NET) and Squamous Cell Carcinoma
(SCC) of the esophagus both in the same
tissue section stained with H & E stain).
Fig. 4-3: presents the neuroendocrine tumor
& carcinoma (NET or NEC) on the left and
squamous cell carcinoma (SCC/on the right)
of esophagus with H & E staining reactivity.
623
For some of the staining options in
neuroendocrine glands refer to chapter
three (3) of this textbook part five. The
following is two types of H & E staining
techniques for an example, one regressive
and the other is the progressive.
Histology:
Routine
Hematoxylin
and
Eosin Staining
The
Progressive
stain is shorter and
is
the
most
frequently
used
method
on
automated staining
instruments.
The Regressive stain is
longer, uses an acidalcohol differentiator
and is the most
frequently used manual
method.
Progressive
Method for
Sections
Xylene
Substitute
Xylene
Substitute
Xylene
Substitute
Absolute
Alcohol
Absolute
Alcohol
95% Alcohol
Regressive H&E Method
for Paraffin Sections
Xylene Substitute 3
minutes
Xylene Substitute 3
minutes
Xylene Substitute 3
minutes
Absolute Alcohol 2
minutes
Absolute Alcohol 1
minute
95% Alcohol
1
minute
70% Alcohol
1
minute
DI Water
1
minute
Hematoxylin*
6-8
minutes
Tap Water
45
seconds
Acid/Alcohol
30-45
(Differentiator)* seconds
H&E
Paraffin
3
minutes
3
minutes
3
minutes
2
minutes
1
minute
1
minute
70% Alcohol 1
minute
DI Water
1
minute
Hematoxylin* 2-5
minutes
Tap Water
45
seconds
Bluing Agent 30
seconds
to
1
minute
Tap Water
1
minute
Tap Water
2
minutes
Eosin
Y 20-40
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* Use either Mercury
Free
Harris
Hematoxylin, or Gill II
Modified Hematoxylin,
**Use
Eosin
Y
Alcoholic, 1% PDC,
*
DI Water
Bluing Agent
DI Water
Tap Water
Eosin
Alcoholic**
95% Alcohol
Y
Absolute Alcohol
Absolute Alcohol
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Running
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Routine
immunohistologic
stains in Histopathology
The
first
action
in
immunohistochemistry (IHC) is the
preparation of the tissue and its fixation.
Preservation needs a prompt and careful
preparation with adequate tissue fixation.
This must be sufficiently fixed so that does
not affect subsequent procedures for
antibody binding and conjugation. A
universal fixation technique does not exist
and in general paraffin fixation may be
an ideal fixative. The development of
antigen
retrieval
techniques
and
microwave treatment and availability of
the corresponding antibodies are new way of
624
quality control of the process and may
demand careful manipulation.
The
most
common
fixatives
for
immunohistochemistry include, 1) 4%
paraformaldehyde in 0.1M phosphate
buffer, 2) 2% paraformaldehyde with 0.2%
picric acid in 0.1M phosphate buffer, 3) PLP
fixative: 4% paraformaldehyde, 0.2%
periodate and 1.2% lysine in 0.1M
phosphate buffer, 4) 4% paraformaldehyde
with
0.05%
glutaraldehyde
(TEM
immunohistochemistry). Under normal
conditions some antigens may be lost due to
fixation or the processing even in a very
small amount of aldehyde fixatives,
therefore the tissue should be rapidly fresh
frozen in liquid nitrogen (vide supra) and
cut by the cryostat microtome without
infiltrating with sucrose. The section must
be kept frozen at -20°C or lower until
fixation with cold acetone or alcohol.
Subsequent to fixation, we may stain it
with standard staining protocols with
immunocytochemistry
stains
and
procedures.
The processing and sectioning may be
done by fixation usually by formalin and
conventional
paraffin
sections
and
impregnation for most antigens with the
current date antigens retrieval technique.
As mentioned some antigens may not
process and survive by these normal
fixations and paraffin sectioning and
infiltration, thus can be done by frozen
section however this methods has the
following disadvantages:
1) Poor morphology
2) Poor
resolution
at
higher
magnifications
3) Special storage needed, and
4) Limited retrospective studies and
cutting difficulty over paraffin
sections.
In this regards vibratome [definition –
Vibrotome.com reference 21: A vibratome is
an instrument that is similar to a microtome
but uses a vibrating razor blade to cut
through tissue. The vibration amplitude, the
speed, and the angle of the blade can all be
controlled. Fixed or fresh tissue pieces are
embedded in low gelling temperature
agarose. (Some have had success without
using the agarose to embed..) The resulting
agarose block containing the tissue piece is
then glued to a metal block and sectioned
while submerged in a water or buffer bath.
Individual sections are then collected with a
fine brush and transferred to slides or
multiwell plates for staining/ Fig. 4-4 shows
a contemporary virotome] may be used for
some sectioning which as the advantages of
no high heat required or no organic solvent
is needed, these can destroy the antigen. In
addition the vibratome will not affect the
tissue morphology and in comparison
with freezing it may prevent constant
freezing and thawing of the tissue sections
and thus help preservation of morphology.
Vibratome sections are often used for floating
immunostaining, especially for preembedding EM immunohistochemistry.
The following are the disadvantages of
vibratome:
1. The sectioning process is slow and
difficult with soft and poorly fixed
tissues, and
2. Chatter marks or vibratome lines
are often appeared in the sections.
part is whole mount
preparation, this include small tissues
sections (5 mm thick) as whole mount. The
The
last
625
advantage is that in whole mount the three
dimensional information is possible without
reconstruction from the section. The
disadvantage may be:
1. antibody penetration may not be
complete in the tissue, resulting in
uneven staining or false negative
staining
Nevertheless the use of Triton X-100 or
saponin treatment routinely for whole
mount immunohistochemistry may
enhance penetration of the antibody.
Fig.4-4: A cryogenic equipment preparation
for tissue sections, note the virbrotome in
the photo operates on the bases of vibrating
razor blade to cut tissues for fixed or frozen
sections. More detail in the text.
Immunohistochemistry uses the properties
of antigen and corresponding antibodies to
screen the tissue sections with application of
conjugate stains as cited before in
histochemistry. Tissue antigens of breast
pathology and or neuroendocrine antigens
(and other tissues/vide infra) and or
neuropathologic proteins as protein gene 9.5
or chromogranin A or synaptophysin and
intermediate filaments may be used as
antigen and the antibody to these antigens.
These can be conjugated to test system dye
applied to tissue may specifically stains the
target antigens of the labeled/stained
tissue.
These
immunohistochemical
markers may include the following tissues:
breast, with markers such as estrogen
receptors progesterone receptor; or liver as
in CAM 5.2 (Cell Adhesion Molecule), AFP
(alpha Feto-Protein), HBsAg (Hepatitis B
Surface Antigen); or thyroid such as
thyroglobulin (as in papillary and follicular);
and calcitonin; and prostate as in PSA
(Prostate Specific Antigen), PAP (Prostate
Acid Phosphatase), Leu 7; or in ovaries with
such markers as estrogen and progesterone
receptors; or lungs as in CEA
(Carcinoembryonic Antigen). In addition,
colorectal antigens as CEA and CK20
(Cytokeratin for Bladder Cancer) are others,
also other antigens such as the
neuroendocrine and neurological markers
cited above. Immunohistochemistry is
accurate and sensitive in detecting the
labeled antigen with antibody and on-going
bases and has been utilized in
histopathology for screening the antigens
in the tissues sections. Breast antigens and
marker such as cytokeratines and
antibodies to these antigens are now
routinely been used in breast pathology of
many tumors and malignant masses in
variety of tissues (which are non-epithelial
in nature) such as synovial sarcoma,
Schwannomas, glial tumors and malignant
melanoma,
and
some
lymphomas.
Antibodies such as CAM 5.2 (Cell
Adehision
Molecule
for
Cervical
cancers/Pan Cytokeratine) and AE1 (Anti
Epidermis Antibody) and or AE3 (or
626
EMA/Epithelial Membrane Antigens),
or breast specific markers also myoepithelial
markers (Dewar et al. as examples S100
protein or antigen, basement membrane
markers, or smooth muscle actin) may be
used in detection of the corresponding
antigens in the afflicted tissues: example
consists of AE1 for epithelial tissues as
breast epithelial tissues. The cocktail of
these antibodies has usefulness in these
detection systems. Other protein of interest
specific for tissues is NSE [Neuron
Specific Endolase (not specific for
neurons)] and its antithetical antibody with
other tissue antigens are purified and uses in
detection of routine immunohistochemistry.
The availability of these antibodies
screening of cells and tissues are in wide
practical with immunohistochemistry and so
many of them has been recovered and
manufactures
by
pharmaceutical
industries, the list of these antigens are
quite long and is out of the scope of this
section. Figure 4-5 shows an epidermal
adenosquamous carcinoma positive for
cytokeratin
by
immunohostochemical
staning with cytokeratin 7 antigen.
Fig. 4-5: a positive cytokeratin in a
epidermal adenosquamous carcinoma, note
the squamous cell tumor with keratinization
and gland formation in goblet cells (goblet
cells show no staining in this silde with
cytokeratin 7) and stromal response, the
immunohistochemical
staining
with
cytokeratin 7 shows a prominent staining of
cytoplasmic and nuclear staining.
These antibodies can be as well used in
conjunction with enzymes and radionuclei
and
fluorescent
techniques (as
fluorescein dyes) to visualized specific
tissues substituents and therefore detect and
diagnose variety of tumors, malignancies
and anomalies of the ablated tissues
sections. Immunohistochemistry also make
uses of enzymes such as ALP, ACP or ALT
and
AST
(Alanine
&
Aspartate
Aminotransferase) or hormones as FSH
(Folicule stimulating Hormone), glucagon,
etc. (such as the Immunoperoxidases/IPO).
This is to investigate the histopathology of
these tissue organs with corresponding
hormone, cytokines, and lymphokines
production in situ. The used of
radioisotopes and radionucleotides
(radiolabelled) is been utilized in such as
settings. The use of cocktails or
combinations of these methodologies has
been warranted in tissues section screening
and detection in immunohistotechnology
and immunocytotechnology. For more
detail and elaborate discussion of the
immunohistechnology staining methods
refer to external other sources or the
references at the end. The following (table
4-1) list is an elaborate list of antigenantibody
markers
used
in
immunohistochemical
and
immunocytochemical staining (Bacroft et
al):
Table 4-1: the most up-todate immunohistotechnoloic
627
and immunocytotechnologic
tissue marker in Pathology
Alpha-1
c-erbB-2,
Her
monoclonal
c-erbB-2, Her 2, (FISH) Breast Cancer
CEA (Carcinoembryonic Adenocarcinoma,
Antigen), monoclonal
metastatic carcinoma
unknown primary
Histiocytic marker
Antichymotrypsin,
CD1a, monoclonal
Thymic
T-cells,
thymoma, Langerhans
cells
CD2, monoclonal
Lymphoma/leukemia
typing
CD3, monoclonal
Lymphoma/leukemia
typing
CD4, monoclonal
Lymphoma/leukemia
typing
CD5, monoclonal
Thymic carcinoma
CD7, monoclonal
Lymphoma/leukemia
typing
CD8, monoclonal
Lymphoma/leukemia
typing
CD10, monoclonal
Lymphoma
typing,
metastatic carcinoma
unknown primary
CD15, monoclonal
Lymphoma
typing,
mesothelioma
vs.
adenocarcinoma
polyclonal
Alpha-Synueclein
2, Breast Cancer
Alzheimer Disease
ACTH
(Adreno Pituitary tumors
Corticotrophic
hormone), polyclonal
Actin Muscle (HHF35), Smooth and Skeletal
monoclonal
muscle
Actin Muscle, smooth, Smooth muscle
monoclonal
AFP
(Alpha- Hepatoma, Germ cell
Fetoprotein), polyclonal tumors
ALK-1, monoclonal
ALK-1
lymphoma
Ber EP4, monoclonal
Mesothelioma
Adenocarcinoma
vs
B72.3, monoclonal
Mesothelioma
adenocarcinoma
vs.
BCL-2, monoclonal
Lymphoma
typing,
soft tissue tumors
CD20, monoclonal
Lymphoma/leukemia
typing
BCL-6, monoclonal
Lymphoma typing
CD21, monoclonal
CA19-9, monoclonal
Metastatic carcinoma
unknown primary
Follicular
cells,
typing
CA-125, monoclonal
Mesothelioma
adenocarcinoma
CD23, monoclonal
Lymphoma/leukemia
typing
CAm5.2
Adenocarcinoma
CD30, monoclonal
Lymphoma typing
Calretinin, monoclonal
Medullary
thyroid
carcinoma
CD31, monoclonal
Vascular
differentiation
Calcitonin, polyclonal
Medullary
thyroid
carcinoma
CD34, monoclonal
Calponin
Smooth
muscle
differentiation,
myoepithelial cells
Soft
tissue
tumor
classification,
leukemia typing
CD43, monoclonal
Lymphoma/leukemia
typing
CD45RB, monoclonal
Recognition
of
lymphohematopoetic
tumors
C-Kit,
monoclonal
positive
vs.
CD117, Gastointestinal
stromal tumors mast
cells
dendritic
lymphoma
628
CD45RO, monoclonal
Lymphoma
typing
(same as UCHL-1)
CD56, monoclonal
Lymphoma
typing,
neuroendocrine
differentiation
CD57, monoclonal
Lymphoma
typing,
neuroendocrine
differentiation
CD68, monoclonal
Histocytic/
myeloid/monocytic
marker
Estrogen receptor
Breast
cancer
prognostic
marker,
metastatic carcinoma
unknown primary
Factor
VIII-Related Megakaryocytic
Antigen, polyclonal
marker,
endothelial
marker
Factor XIII-A-subunit, Dermatofibroma
polyclonal
DFSP
Fascin, monoclonal
vs.
Hodgkin's Lymphoma
FSH
(Follicle Pituitary tumors
Stimulating Hormone),
polyclonal
CD79a, monoclonal
Lymphoma/leukemia
typing
CD99, monoclonal
Soft
tissue
classification
CD123, monoclonal
Lymphoma/leukemia
typing
CD138, monoclonal
Plasma Cells (also
stains many epithelial
tumors)
Chromogranin,
monoclonal
Neuroendocrine
differentiation
Clusterin, monoclonal
Anaplastic Large Cell
Lymphoma
HCG (Human Chorionic Choriocarcinoma
Gonadotropin),
polyclonal
CMV
(Cytomegalovirus),
monoclonal
Cytomegalovirus
infection
Herpes Simplex Virus Herpesvirus I and II
Type I, polyclonal
infection
Cyclin D1, monoclonal
Mantle cell lymphoma
Herpes Simplex Virus Herpesvirus I and II
Type II, polyclonal
infection
tumor
Cytokeratin
monoclonal
7, Metastatic carcinoma
unknown primary
Cytokeratin
monoclonal
20, Metastatic carcinoma
unknown primary
Desmin, monoclonal
Smooth and skeletal
muscle differentiation
EBER (ISH)
Epstein Bar Virus
E-Cadherin,
monoclonal
Lobular vs. Ductal
breast carcinoma
EGFR, monoclonal
Sweat
gland
carcinoma, squamous
tumors
EMA
(Epithelial Metastatic carcinoma
Membrane
Antigen), of unknown primary,
monoclonal
Lymphoma typing
Epstein
(CSI-4)
Barr
Virus Epstein Bar Virus
Gastrin, polyclonal
Gastrinomas
GFAP (Glial Fibrillary Glial tumors, salivary
Acid
Protein), gland tumors
monoclonal
GH (Growth Hormone), Pituitary tumors
polyclonal
Glucagon, polyclonal
Pancreatic
tumors
endocrine
Hepar
Hepacellular
Carcinoma
HHV-8
Kaposi Sarcoma
HMB-45
Melanoma
marker,
angiomyolipoma
IgA (Alpha Chains & Plasma
Secretary
subclassification
Component),polyclonal
cell
IgG (Gamma Chains), Plasma
polyclonal
subclassification
cell
IgM
(Mu
polyclonal
Chains), Plasma
subclassification
cell
Plasma
subclassification
cell
IgD
Insulin,
(GP)
monoclonal Pancreatic
tumors
endocrine
629
Keratin, AE1
Epithelial Tumors
Pax 5, monoclonal
Keratin, AE3
Epithelial Tumors
Keratin 903
Prostate marker
Placental
Alkaline Germ cell tumors
Phosphatase, polyclonal
Kappa light
polyclonal
chains, Plamsa cell clonality
Ki67, monoclonal
Proliferative
fat cells
fraction,
Lambda Light Chains, Plamsa cell clonality
polyclonal
Laminin
Skeletal muscle
LH
(Lutenizing Pituitary tumors
Hormone), polyclonal
Lysozyme
(Muramidase),
polyclonal
Myeloid
and
histiocytic marker
MART-1
Melanoma,
adrenal
cortical, sex cordstromal tumors
Mast Cell Tryptase
Mast Cell Tumors
MLH-1
Colon
Adenocarcinoma
PMS2
B-Cell Lymphoma
Colon
Adenocarcinoma
Progesterone Receptor, Breast
monoclonal
marker
Prolactin, polclonal
prognostic
Pituitary tumors
PSA (Prostate Specific Prostate marker
Antigen), monoclonal
Racemase
Prostate marker
S100, monoclonal
Spitz nevus
Seratonin, monoclonal
Carcinoids
Somatostatin,
polyclonal
Pancreatic
tumors
Spirochete
Spirochete
Synaptophysin,
monoclonal
Neuroendocrine
differentiation
TCRbf1
Lymphoma typing
TIA-1
Lymphoma typing
endocrine
MLH-2
Colon
Adenocarcinoma
TDT
Lymphoma/leukemia
typing
MLH-6
Colon
Adenocarcinoma
TTF-1
Myogenin, monoclonal
Rhabdomyosarcoma
Lung
&
thyroid
marker, also some
neuroendocrine
Myosin
Skeletal muscle
Thyoid carcinoma
MUM-1, monoclonal
Lymphoma Marker
Thyroglobulin,
monoclonal
Myeloperoxidase
Acute Leukaemias
N-Cadherin
Metastatic caricnoma
unknown primary
Neurofilament
160 Neural tumors
(NN18), monoclonal
NSE (Neuron Specific Pancreatic
Enolase), monoclonal
tumors
endocrine
p16
Cervical
dysplasia
(correlates with high
risk HPV)
p53, monoclonal
Breast Carcinoma
Pan Keratin AE1/AE3
Epithelial Tumors
Parathyroid Hormone Parathyroid Tumors
(PTH), polyclonal
TSH
(Thyroid Pituitary tumors
Stimulating Hormone),
polyclonal
Vimentin, monoclonal
Metastatic Carcinoma
Unknown Primary
*The source of the list: The Histology and
Immunohistochemistry
Laboratory
Department
of
Pathology,
The University of Texas Health Science
Center at San Antonio
630
Immunohistochemistry
staining and markers (IHC)
of Lymphomas
There are two broad classifications of
lymphomas by WHO (World Health
Organization)
with
the
lymph-node
involvement. The first classification is
Hodgkin’s Lymphoma (HL) and the
second classification is non-Hodgkin’s
lymphoma,
Chapter
under
lymphoproliferative disorder has cited
both of these lymphomas in part two of
diagnostic hematology in this textbook
however we will consider these topic on the
bases of immunohistochemical staining
which had been partly included in diagnostic
hematology section. WHO further classify
Non-Hodgkin’s Lymphoma (NHL)/
(Martin et al) according to the maturity
(differentiated)
and
immaturity
(undifferentiated) of the cells lines and the
lymphatic tissues itself (cells of origin)
using the basis either on immune cells
such as T cells, B-cells and or Natural Killer
Cells (NK).
As with specimen collection and transport
we have to understand the correct handling
of the specimen in the laboratory on its
arrival in the laboratory. This is crucial to
successful sample reporting. Although many
diagnoses can be reached using routinely
processed tissue, ancillary tests are essential
for some diagnoses such as Burkitt
lymphoma.
Tissue Fixation: Lymph node specimens
larger than 0.5 cm in diameter need to be
sliced on arrival in the pathology
department, this allows proper fixation. The
tissue sample in paraffin blocks must be 3
mm thick. If possible, one section should be
placed in each cassette since multiple pieces
make immunostaining more difficult. We
should be certain that the slices of lymph
node remain flat, incorporate sponges into
the cassettes to help prevent folding. The
blocks need to be fixed for 24-48 hours; less
than this leads to poor preservation of
cytological detail and can make the tissue
uninterpretable. Standardization of fixation
makes immunochemistry more reliable,
since heat and protease recovery time will
be similar. Prolonged fixation makes
immunochemistry more difficult and
recovery of DNA (Deoxy Ribonucleic Acid)
from paraffin blocks unreliable for
cytogenetic and molecular studies.
Flow Cytometry
If the sample is to be analyzed promptly, a
dry fresh sample should be provided to the
appropriate histotechnology laboratory. If
this is not possible due to transport times,
consideration should
be
given to
disaggregating and fixing the sample prior to
transport for analysis.
Bone marrow aspiration and trephine
biopsy
Bone marrow aspiration is useful for
morphology, FC (Flowcytometry), PCR
(polymerase Chain Reaction), FISH
(Fluorescent In Situ Hybridization) and
occasionally
conventional
cytogenetic
analysis if no lymph node material is
available. In spite of aspiration, the value of
bone marrow trephine biopsy in the
diagnosis and staging of lymphoma is well
established (Bartl, et al, Burkhardt, et al,
Munker, et al). The trephine biopsy core can
preferably be taken from the posterior iliac
crest with multiple sections taken from
various dimensions. As in collection and
preparation of bone marrow trefine biopsy
the sample should be collected into 4%
formalin as this fixative allows the
subsequent use of most staining and
631
molecular techniques (Le Maitre, et al). The
sample may then be stored for 72 hours or
more before preparation, sufficient for
samples to be posted to the reporting
laboratory where necessary (Krenacs, et al).
Subsequent preparation will depend on the
preferred method of the laboratory. Bone
marrow trephine (Beakstead et al) sections
should always be stained with H & E and
with a reticulin stains; a Giemsa stain can
also be helpful. Each slide should be
examined initially for cellularity, the
trephine core being the best sample for
assessing this. Light microscopy alone
cannot be relied on to distinguish nonneoplastic follicles or lymphoid hyperplasia
from lymphoma infiltration. However, it is
possible, when no other tissue is available,
to diagnose lymphoma solely based on a
trephine biopsy specimen, provided the
appropriate immunostaining protocol. In this
regard, immunostaining should be requested
as a panel of antibodies rather than
individual tests so appropriate comparisons
can be made. It can be performed by FC,
(Flowcytometry) which readily identifies
single cell populations.
Paraffin/ plastic section immunochemistry
has the advantage that the histologically
abnormal cells can be seen to express or lack
a particular marker as in phenotyping. If all
tissue blocks are similar, immunostaining
needs to be performed on one block only,
otherwise blocks need to be selected to
demonstrate all suspected diagnoses in a
patient; i.e. the presence of diffuse large Bcell lymphoma on a background of follicular
lymphoma. When selecting panels for
immunohistochemistry
and
immunocytochemistry it is important to
include antibodies that are expected to give
negative as well as positive results.
In addition, lymphoma diagnosis may be
made from a number of different specimens
depending on the presenting clinical
features. Histotechnologist in the
histology laboratories may expect to receive
lymph nodes, bone marrow aspirates,
trephine biopsy cores and peripheral blood
for processing of tissue block and section for
diagnosis by the pathologist as well as other
fluids such as Cerebrospinal Fluid (CSF),
ascitic fluid and pleural aspirates. In other
hospitals, tissue samples may be received by
the cellular pathology department. The
number of pathological tests needed for the
precise diagnosis and classification of
hematological malignancies means that
whole lymph node samples are preferred,
rather than needle cores or Fine Needle
Aspirate Biopsy (FNAB) cytology. Core
biopsy is useful in the diagnoses of tumors
which are not accessible such as those in the
retroperitoneum but the diagnosis of tumors
where the architecture is important,
especially
low
grade
Non-Hodgkin
Lymphoma (NHL) (Martin et al) and
Nodular Lymphocyte Predominant
Hodgkin Lymphoma (NLPHL) can be
difficult from these samples. The easiest
way to achieve ideal/optimal results is to
arrange for lymph nodes to be immediately
submitted fresh to the laboratory. This
allows not only collection of fresh tissue
samples for immediate analysis, but also
optimum fixation for paraffin/ plastic
embedding and processing, and the
preparation of imprint cytology specimens
(for detail tissue processing refer to the
beginning of the chapters 3 & 4). Staining
with different immunochemical stains
and conjugation with different batteries of
antibodies may help visualization of the
tissue labeled/tagged sections. As
mentioned in the previous section the
markers and antibodies are cited in the long
comprehensive list for different tissues.
632
In general on the level of tissue anatomic
alteration and pathology these may consist
of change in morphology of the lymphocytes
or the affected cells of nodal origin from
normal tissue structures such as B-cell
follicles, mantle or peripheral tissue
textures or on T-cells intra-follicular region
or sinus zones. All aspects of cell
morphology may be considered in this so
that a correlation may be made to diagnosis,
such as cytoplasmic texture and
inclusions or nuclear appearance, pigments
and cytoplasmic and nuclear chromatic
appearance may be clue to the right
diagnosis.
Parameters such as cell size as large or small
or polychromatic or monochromatic cell
population may be used in diagnosis as well.
The occurrence of the proliferation is
generally due to arrest of differentiation
stages of cell and subsequent proliferations
at different stages of development. In
immunohistochemistry of the lymphomas
we chose a panel of markers based on
morphologic differential diagnosis (no
single marker is specific): these consist of
Leukocyte Common Antigen (LCA),
B-cell markers (CD20 and CD79a), T-cell
markers (CD3 and CD5/cluster of
differentiation 3 & 5) and other markers
etc. The beginning IHC panel may include
antibodies against/to B-cell (CD20) and Tcell (CD3) antigens; κ and λ light chains (if
numerous plasma cells are present); and
D45, CD15, and CD30, this is when large
dysplastic cells are seen. Based on
morphology, if there is changes/alteration of
B-cell zones, antibodies against some of the
markers as CD5, CD10, CD23, etc. could
also be useful primarily characterize the
staining process for identification. If interfollicular areas expanded and a T-cell
lymphoma is a consideration, antibodies
against CD2, CD4, CD5 and some other
may identify the subset distribution and if
pan–T-cell antigens are abnormally lost
(US-Canadian division of international
academy of pathology).
B-cell and T- cell markers and follicular
dendritic cell markers can distinguish
conditions such as T- cell and B-cell
leukemia, T-cell and B-cell lymphoma, or
small cell carcinoma and urothelial
carcinoma and rarely endometrial and
breast cancers. Angioimmunoblastic T-cell
lymphoma may be a cause of follicular
dendritic cells proliferation.
With regards to Immunohistochemistry
staining procedures is less sensitive than
flowcytometry, due to FC accurate and
precise identification and sorting of tissue
and cells. The immunoglobulin light chains
may be stained to differentiate abnormal
clonal cell populations.
Hodgkin’s Lymphoma (HL) is diagnosed by
screening for Reed-Steenberg cells
(RS)/(refer to diagnostic hematology chapter
on lymphproliferative disorders, part two of
the textbook). The parameter to consider is
the cells, inflammatory background and
immunophenotyping of the RS cells.
Figure 4-6 shows Reed-Sternberg cells
stained with immunocytochemical stains in
diagnosis of HL.
633
1. Solving
common
diagnostic
dilemma,
2. Tumor typing and subsequent
diagnosis, and
3. Analysis of prognostic markers, i.e.
ER (erstrogen).
Therefore I will expand the idea of
immunstaining of tissues of some classes of
breast pathological entities with their
relevant antigens and antibodies utilized in
here further below, the examples are (I start
with fist category the dilemma):
Fig. 4-6: Reed-Stenberg cells (four at the
center) in Hodgkin’s lymphoma by H & E
staining with 400x magnification, the
section
has
been
treated
with
immunochemical stain for CD30 positive:
source lung tissues.
Immunohistochemistry
staining and markers
Breast pathology
of
1. Benign
tumors
vs.
malignant
tumors,
2. Epithelial proliferations, and
3. In situ vs. microinvasion.
The second one tumor typing may be
divided into the following types:
1. Typing of tumor (e.g. ductile vs.
lobular, or basal vs. myoepilthelial),
2. Metastases of lymph-nodes, and
3. Demonstration of epithelial cells in
necrotic materials, and others, etc.
(Dewar et al & Bancroft et al)
It is clear that H & E staining of the breast
tissue section although would be reliable for
the diagnosis nevertheless the diagnosis
could not be based solely upon it. However
one of the new although accurate and
precise methods of diagnosis of breast
pathology may be obtained from
immunohistochemical evaluation of
breast tissue or acquired breast cells in
immunocytochemistry (we will not focus in
here
on
immunocytochemistry).
Immunohistochemical approach can respond
to the queries that might arise from H & E
staining. The main purposes of the
immunhistochemical staining are:
The third category analysis of prognostic
markers (i.e. ER)
1. Receptor studies and assessment
of these such as ER/PGR and Her-2
receptor(s)
2. FISH testing for Her-2 (This will not
be covered as being solely a clinical
genetic specialty).
There are several antigens as list continues
to expand we may have the category of
immunostains
to
use
in
immunohistochemistry and breast pathology
which are popular in approach, these are
(with many others have been cited later):
634
1. Myoepithelial markers (CK 5 &
6 and P63)/ these are benign in
orign),
2. Lobular vs. ductile (as Cadherin)
and,
3. Receptors such as ER (estrogen),
PGP and Her-2 receptors.
For the above details refer further below
(vide infra). We receive some different
specimen and tissue types of breast tissues
sections in histothechnology lab for
breast pathology these include, preoperative
specimen as FNAB and core biopsy, and
the next are the operative tissue specimen
such as excision biopsies, mastectomies and
lymph-nodes. Investigation may be carried
out for benign and malignant lesions
such as fibroadenoma or other fibroadenoma
tumors, also papilloma, or inflammations,
abscesses, necrotic foci, radiation injuries or
mammary duct fistula, or so on; for
malignant lesions with grading (cancer in
situ) we have ductal and lobular carcinoma
in situ (not invasive), invasive carcinoma,
invasive lobular and tubular carcinoma, and
invasive mucinous carcinoma (MUC-1).
As
with
fibromatosis,
immunohistochemical staining uses for core
biopsy include beta-catenin which is
supportive for diagnosis. Staining may be
patchy: for differential diagnosis a positive
result with beta-catenin which may be
positive rarely and uncommonly in
differentiation of malignant tumor types.
This can be found positive in fibromatosis.
To detect myoepithelial cells in the
mammaries we utilize immunostain
markers of high specificities and
sensitivities such as smooth muscle myosin
heavy chains and calponin P75, P63, Pcadherin, basal cytokeratin, maspin and
CD-10 these antigens may be detected with
corresponding antibodies with an ease of
interpretations: these markers may be
positive or negative in carcinoma in situ.
These may cross react with reduced
expression of myoepithelial cells in
borderline conditions as with in situ
carcinoma (Dewar R. et al). As mentioned
earlier myoepithelioma markers are benign
in origin and may be distinguished by such
immunohistochemcial marker as cytokeratin
19, 18 and 8 are diagnostic (vide infra).
Immunological and histochemical
markers for staining methods such as
cytokeratins (19, 18 and 8) can be found
in epithelial tumors these antigens may have
antibodies such as CAM 5.2, AEI and AE3
etc. these can be used in such conditions as
myopithelioma of breast and these
cytokeratins may as well be found as a result
of cross reactivity with non-epithelial
derived tumors [as cited in routine
immunohistologic
stains
used
in
histotechnology and histopathology section
under this chapter (4)]. Nonetheless, other
markers such as epithelial membrane
proteins (EMP/ its use have been reduced
during recent time) may provide a good
diagnostic decision in adenocarcinoma
(Fig. 4-7) and shows positive expression.
Antigens such as breast specific markers
as Gross Cystic Disease Protein Fluid 15
(GCDPF 15) is found in fibrocystic
changes as well and is an excellent marker
for detection of such changes.
Still with other antigens we may include
Smooth Muscle Actin (SMA) and S100
protein. SMA may be detected in skeletal
muscles, smooth muscles and cardiac
muscle. For S100 protein which is
detected in variety of tissues tumors such as
635
schwannoma,
epithelioma (islet of
Langerhans cells of epidermis), melanoma,
also in heart muscles, chondrocytes and
lipocytes and histiocytes, this marker can be
used in breast pathology as well as in other
tissues and may be detected and originate in
breast tissue sections afflected with the
corresponding tumors or conditions.
Other antigens of importance are calponin,
caldesmin, cytokeratin 14 and 5, caspin,
and P-cadherin. Basal membrane proteins or
components such as laminin or collagen IV
with their correspondent antibodies may be
utilized in investigation of differentiation in
situ epithelial tumors versus invasive
carcinomas, these are positive in former
condition due to possession of intact
epithelial membrane (in situ). In addition
neuroendocrine markers, neuronal specific
antigens and proteins (markers) including
other markers as chromogranin A and
sypatophysin that had been cited in the
previous preceding sections may play a
significant role in detection of these type
tumors of breast and its pathology (vide
supra/Bancroft, et al.).
Fig. 4-7: presents Epithelial Membrane
Antigen (EMA) staining of a case of
adenocarcinoma of the breast in a tissue
section.
In respect to the above markers I have to
explain little about the MUC-1 a
glycoprotein (or MUC 2 & 3 & 8 and
mucinous carcinoma), as a mucinous
marker, we have a molecule such as
epithelial membrane mucins (MUC2/glycoproteins, etc.) which once present
can be an indication of mucinous
carcinomas of mammaries. These are
trans-membrane proteins that are the
components of breast and other epithelial
cells that produce milk during lactation.
These may be mucins present in glandular
epithelial cells and mucins detections
with corresponding antisera led to the
discoveries of antigens such as EMP, MM or
PEM (Polyepithelial Mucins) and episialin.
These mucins have thought to be produced
in the breast glandular epithelial cells. These
MUC-1 glycoprotein and their 20 amino
acids core antibodies (prepared antisera)
may bind to the breast cancers antigens
showing homology. Therefore MUC-1 and
other mucins may play a role in breast
cancer pathology. Other antigens of interest
may be growth factors and their receptors:
such as epithelial cell growth factors
(EGF/a Epithelial Peptide Growth Factor)
which may function as autocrine or
juxtacrine growth factors in breast epithelial
cells and are regulated by estrogens (as a
hormone). Tyrosine kinase growth
factor receptors such as Her-2, Her-3, etc,
have been found to play role in breast cancer
and its induction as well. Therefore may be
used in breast cancer detection (the list of
markers mentioned in early this chapter may
be used to investigate some of the tumor
markers mentioned in this section.).
636
Histochemistry by enzyme
applications
(Beckstead et al & Burstone et al)
It is currently becoming popular and
mandatory to study major part of the tissue
block or sections arriving at laboratory by
stains with either enzyme application in
histotechnology
or
immunohistochemical stains however
still tissue processing such as fixation,
dehydrating, clearing and impregnation with
paraffin or MMA and GMM has important
applications in histopathology. With this
application (paraffin impregnation) we can
use enzyme or immunstains.
With enzymes processing it still would be
similar to doing fixation of the slide or the
tissue with formalin and applying alcohol or
acetone for dehydration and clearing along
with impregnation with paraffin, MMA or
GMM. The sections that are 2 micron thick
(depending on the type of tissue) must be
incubated at 4°C with peroxidase, or
naphthol
AS-D
chloroactetate
esterase, alpha naphthol butyrate esterase
(positive in myelomonocytic, monocytic
leukemia and weakly in megakaryoblastic
leukemia/ these are specific and non-specific
esterases respectively) and alkaline
phosphatase (ALP) or acid phosphatase
(ACP) with ancillary reactions. This may be
used to investigate for different purposes
such as hematologic and other cancers or
leukemia. In 48 hours the result will be
investigated and read according to the
protocol. This method can be used in focal
lesion studies of bone marrow such as
inflammation and neoplasia or similarly
myelofibrosis of the bone marrow may show
increase number of alkaline phosphatase.
Other enzymes may also be used aside from
the above enzyme to detect other disorders
(Beckstead. JH et al.). In regard to
enzymatic methods we also can make use of
aceytlcholenstarease enzyme E method
in fresh frozen section to localize the AchE
in the neuronal tissues.
Development of techniques in enzyme
histochemistry would enable the pathologist
to search for the correspondent enzyme in
the tissue sections and observe their
distribution pattern and their localization
in a particular tissue biopsy. The aim of
enzymes histochemistry is to study the
effects of various substances as enzymes by
the aid of enzyme labeling of different
tissues to study their effects on the cellular
and tissue level. Respectively enzymes such
as horse reddish peroxidase may be
substituted in enzyme-histotechniques
for fluorescein in immunohistochemistry.
For the definition and pathophysiology of
enzyme refer to clinical chemistry section
part one of this textbook.
Classically enzymes are divided into six
classes:
1)
oxidoreductase,
2)
transfereases 3) hydrolases, 4) lyases, 5)
isomerases, and 6) ligases. And majority
of enzymes lay under these categories. The
following factors may influence the
demonstration of enzymes in tissues: 1)
temperature, this is important for
localization of each enzyme and may need
special attention to the enzymes
surrounding temperature, 2) PH of the
surrounding, 3) inhibitors, these may block
the enzyme activity, 4) activator, this may
promote enzyme activity, and 5) circadian
rhythm: seasonal and daily variation in
enzyme amount may eventually affect the
localization and its detection. As for
detection of enzymes, fresh frozen
637
sections are ideal for localization and
preservation of such substances.
Specimen preparation
In here there may be no optimal fixation
preparation for each particular enzymes and
tissue may be processed either as fixed or
unfixed fresh frozen or paraffin embedded
according to some tissue sections and
staining protocol.
Since
enzymes
are
sensitive
and
denaturable by heat and fixatives the idea
of fresh frozen sections may warrant. In
these, unfixed frozen sections have their
own disadvantages as diffusion of enzyme,
cofactors and activators from their initial site
in the tissue. This may cause false
interpretation of results either false positive
or false negative.
The preparation and cutting may be done by
cryostat microtome as like other tissues and
other procedure mentioned earlier. The
freezing may accomplish either by cryostat
freezer or liquid nitrogen or isopentane
(this is particularly good for muscle tissue).
Aside from fresh frozen sections for
preservation and detection of soluble
enzymes we need to be aware that this may
show diffusion artifacts. In this method there
is less progressive enzyme loss. The
disadvantage of this method (fixed sections)
is deactivation of the target enzyme and
dissolution of certain enzymes in the
fixative. In some method prefixing or post
fixation of the tissues in cold formal calcium
prior to or after cryostat may necessitate.
Frozen sections finally may be method of
choice and tissue may be picked on slide and
stained on coplin jar. The blood or bone
marrow fixation with formal-methanol and
or citrate buffered acetone solution may be
used for slide fixation and tissue section
preparation along with controls to track
false negative or false positive reactions
(due to enzyme diffusion and delocalization
or splitting of activator or the possibility of
presence of inhibitors).
The most common enzymes used in enzyme
histochemistry are: alkaline phosphates
(ALP/tissue and smears), acid phosphatase
(ACP/tissue
and
smears),
ATPase
(Adenine/-nosine
Triphosphatase,
aminopeptidase, phosphorylase, G6PD,
succinic
dehydrogenase,
peroxidase,
NADHase
(Nicotinamide
Adenine
diphosphatase), NADPH along with
naphthol AS-chloroacetate and alpha
naphthol butyrate esterase. The
difference between ACP and ALP is their
PH differences while the reaction is
typically similar (clinical chemistry section
chapter and part one). I will explain few of
these enzymes here with their specificities:
the ALP can be found in areas such as brush
border of convoluted tubular cells of the
kidney and the brush borders areas of
jejunum as well as blood and bone marrow
also we have them in bladder, liver, breast,
adrenal glands and ovaries. It can give a
strong positive reaction (ALP) in bone
tumors (such as Ewing’s sarcoma,
osteogeneic sarcoma) or in monocytic
leukemia and or leukemoid reactions Figure
4-8 shows staining pattern with ALP
enzyme staining. As with ALP we can use
TRAP method (Tartrate Resistance Alkaline
Phosphatase) to screen for the enzyme.
638
and histiocytes stain positive with ACP
staining method therefore may be used as
control (there is no need of control for the
test slide), this is used as a natural internal
control. The purpose of enzyme staining
include incubating medium pH 4.7 - 5.9 for
ACP or 9.0 for ALP the positive enzyme site
indicates inflammatory cells. Methyl green
(methyl green, veronal acetate buffer) may
be used as background counterstain.
Sites of acid phosphatase activity will be
red (indicates necrotizing or inflammatory
myopathy). Background shows a green
color.
Fig. 4-8: ALP enzyme staining of bone
tissue showing red as osteoclast with TRAP
staining
(Tartrate
Resistant
Acid
Phosphatase),
brownish
red
for
chondrocytes and internal cell membranes
stained with ALP immune-enzymatic
staining and bluish green for nuclei with
nuclear staining, this is a normal bone
features.
Equally with ACP, it is found mostly in
disease originating from prostate such as
prostate cancer or benign prostate
hypertrophy (BPH) in these tissue
sections. Some inhibitors such as low level
tartrate, fluoride (uninhibited with formalin
inhibitor) may affect the enzyme. These
enzymes methods need to be used either by
fresh frozen sectioning or by fixed tissue
sections. Both demonstrations of ALP and
ACP need the use of Azo dyes in tissues.
The acid phosphatase stain indicates the
presence of inflammatory cells in the biopsy
as well; acid phosphatase is considered a
marker enzyme for lysosomes. Muscle fibers
in acid maltase deficiency also show an
increase in acid phosphatase. Additionally,
frozen sections of unfixed tissue are
desirable. With regards to ACP, leukocytes
As with the ALP we use this enzyme for
detection of regenerating muscle fibers.
Mechanism includes alkaline phosphatases
hydrolyze
esters
bonds
of
orthophosphoric acid at alkaline pH (see
above). After hydrolysis, the alcohol residue
of the substrate (naphthol AS-BI phosphate)
is reacted with fast red violet to produce a
colored insoluble Azo dye (simultaneous
coupling method): after the two coupling
reaction (not cited) a red Azo dye forms.
Equally with the sections there is no need of
fixation (an unfixed) and a fresh frozen
section is applicable. As with skeletal
muscles staining we must have a positive
and negative control sections because the
blood and vessel walls have small amount of
ALP in them. The stains may be done with
Mayer’s or Harris’ hematoxylin. Site of
enzyme activity shows pink to red colors
with nuclei blue.
As per NADH the enzyme may be used for
demonstration
of
abnormalities
in
mitochondria, Z-band material and
sarcoplasmic reticulum. With NADH
enzyme a NADH diaphorase (a flavoprotein
found in mitochondria to mediate
conversion of NAD) staining techniques
639
may be used and the result for this method
shows sites of enzyme activity to be a
dark purple deposits. Z-band material,
sarcoplasmic reticulum & mitochondria all
react strongly with the method. Normal
skeletal muscle shows a checkerboard
pattern of type I fibers (dark purple) and
type II fibers (light color). Architectural
changes in muscle are indicated with this
procedure including central cores, target
fibers, nemaline rods and targetoid fibers.
Central core disease is when the central area
of the muscle fiber is devoid of
mitochondria and oxidative enzyme activity
however the peripheral zone is normal. The
presence of many target fibers is
diagnostic of a neuropathic disease
process.
Regarding the succinic dehydrogenase,
this is to further identify the source of
NADH diaphorase (sweat induction by this
enzyme/vide infra as in flavoprotein)
activity, because only mitochondria show
positive SDH activity, this stain is used to
demonstrate mitochondrial proliferation
and to distinguish between oxidative and
non-oxidative fibers. As for this enzyme
(SDH) unfixed fresh frozen tissue
sections are preferred. Staining for SDH do
not need control sections as it has naturally
an internal control. Site of ADH activity
shows blue color and the only inclusions
stained are mitochondria. In this regards
oxidative type (I) and non-oxidative type (II)
fibers show dark and light fibers
respectively.
As with naphthol AS-D acetate esterase
a fresh frozen tissue or fixed cryostat
sections may be cut and suited for
preparation of the enzyme staining
procedures: one of the enzyme staining
mechanism include the enzyme may
hydrolyze indoxyl acetate substrate to
free indoxyl. This soluble product of
enzyme hydrolysis transform into insoluble
indigo blue dye. This indigo blue dye
trandformation is attained by the addition of
ferrocyanide or potassium ferricyanide
oxidizers. This will result into indigo blue
dyed tissue sections with good localization.
The sources of enzyme include organs such
as kidney and liver. This esterase contains in
lysosomes of leukocytes and identifies
granulocytes (eosinophils & basophils) to
distinguish leukemia and granulocytic
sarcomas found in acute myeloid leukemia
known as chloromas. The enzyme
hydrolyses esteric bonds (by esterase), they
could be specific or non-specific. This
esterase AS-D acetate is a specific enzyme
and can be performed on paraffin sections
(this is a rare enzyme capable of performing
on paraffin embedding). As in alpha
naphthol acetate esterase can react with
pararosaniline to give an Azo dye reaction
resulting into Azo dye color. For fixing the
section we can use alcohol (methanol) or
formaldehyde or EDTA for decalcified bone
studies. Paraffin sections may be processed
to the slide and dried overnight in an
incubator or room temp. Subsequent to
staining with hematoxylin the nuclei will
be blue. The positive slide may indicate
granulocytes, mast cells and myelogenous
leukemic cells specific for myeloid series.
The alpha naphthol acetate esterase (αnaphthol acetate esterase) is a nonspecific
esterase stain is most useful for
differentiating between different types (I &
II) of atrophy (myopathy) and neurogenic
atrophy (neuropathy); because in neuropathy
denervated muscle fibers stain dark and
in myopathy type II do not. Motor endplates as in neuromuscular junction and
lysosomes in inflammatory cells are also
640
demonstrated. This enzyme hydrolyzes ester
bonds and either specifically or nonspecifically. This can stain marcophages
or histiocytes or other macrophages of organ
and tissue interests such as microglial cells,
or splenic and liver macrophages (i.e.
kupffer cells) and lysosomes, etc. In Azo
reaction the product of hydrolysis of alpha
naphthol with diazonium salt will induce
Azo dye as a dark red brown coloration in
localizing tissue. Source of esterase activity
may be found in kidney, stomach and
jejunum and the liver.
With ATPase, we may identify type I, IIA
and IIB fibers and aids in classification of
myopathies or neuropathic origin of this
enzyme by this tissue marker. In short after
several enzymatic and biochemical reaction
to follow a visible black percipitate of
cobalt-sulfide
will
be
demonstrated
indicating, presence of ATPase in the tissue
section. The screening for ATPase need a
fresh frozen unfixed tissue section cut by the
cryostat application. The above procedure is
PH dependent and the specific PH may
indicate the specific type of the tissue
enzyme.
Last to mention is the modified Gomori
trichrome stain uses for detection of
pathological changes in muscles and
connective tissues with a good staining
properties of muscles and nerves system.
Architectural alterations in muscle fibers
such as nemaline rods or other changes can
differentiate pathological inclusion bodies in
the sarcoplasm (cytoplasm of striated
muscle fiber). Nemaline Rods Cells
Myopathy is a pathological neuromuscluar
disorders that affects muscles and causing
muscle weakness. For a reference to other
enzymes, clinicopathologic properties,
methods and procedures consult one of the
following references cited.
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