CP H58 Histology Competency Manual

Revision 1 | David Muskett
July 18,
2011
HISTOLOGY COMPETENCY MANUAL
HISTOLOGY COMPETENCE
MANUAL
WHAT IS CELLULAR PATHOLOGY
Cellular Pathology is the study of
DEFINITIONS
Specimen
EAST
Biopsy
LANCAS
HIRE
Fixation
HOSPITA
LS NHS
TRUST
Often used interchangeably with sample, describes the material

removed from the body and sent to the laboratory.
Often used to describe a small piece of tissue removed, often for
initial diagnosis
CP/H58 - HISTOLOGY
COMPETENCY
The process of tissue preservation with chemicals. Fixation acts to

MANUAL
prevent the natural processes of cell death, autolysis and
putrefaction.
Autolysis
The process of self digestion within a cell where the enzymes i.e.
auto self, lysis splitting.
Putrefaction
The process of cellular degradation due to micro-organism
Processing
The removal of water from tissue and replacing it with another

substance such as paraffin wax but sometimes tissues are also
processed to other media (e.g. resin).
Request form
The document supplied with tissue samples giving details of the

patient and of the specimen sent. The request form should also
include clinical information.
Reagents
The chemicals and solutions used in the histology laboratory
Haematoxylin &
Eosin
The standard staining method used in histology. It is able to show

good nuclear and cytoplasm detail and gives general tissue
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architecture.
Special stains
A range of stains used to identify specific tissue elements. Usually a

single element of tissue is investigated with special stains such as
fibrin or glycogen, acid mucins or elastin.
Histochemistry
The process of staining cells using the enzymes within the cells to
facilitate the investigation.
Immunocytoche
mistry
A range of antigen antibody related tests where antibodies are

raised against specific tissue elements, which may be nuclear,
cytoplasmic or membrane bound. The antibody reactions are
identified by precipitating a coloured product via an enzyme
reaction at the site of the antibody antigen reaction.
Staining
Human tissues do not look coloured under the microscope by

themselves. Tissue sections require treating with chemicals which
give colour. Some chemicals are coloured and others have colour
when they react with the tissue.
Section
A thin piece of tissue. This is usually one or two cells thick,

approximately 4m thick, so that the tissue is easy to analyse
microscopically. Occasionally there is requirement to examine
material in thinner sections e.g. renal biopsies and material for
electron microscopy or as thicker sections e.g. neuropathology
samples.
Frozen section
A section cut from a piece of tissue which has not been processed
and usually not fixed but has been snap frozen. The tissue freezes
and goes firm. Once hard the tissue may be easily cut with a
cryostat. Frozen sections are sometimes required for rapid real time
diagnosis of patients undergoing surgery or for determining tumour
margin clearance in cases of Mohs micrographic surgery.
Specimen pot
The container in which a specimen is received. It should be labelled

with the details of the patient demographics plus the name of the
clinician performing the biopsy and the sender clinic details. In
addition there should be information on the type of specimen
taken.
Resection
A large tissue sample removed for analysis. It may be a kidney, a

breast, and a length of bowel or a lobe of liver.
Incisional biopsy
A skin biopsy showing some normal tissue and some of the lesion.
It is not a complete removal due to the size of the lesion or its
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HISTOLOGY COMPETENCY MANUAL R1
anatomical position.
Excisional biopsy
A skin biopsy with the lesion completely removed There should be

a margin of normal tissue removed as well.
Microtome
A machine which cuts sections. The basic operation is a knife and a

specimen holding mechanism. The specimen holding mechanism
advances a set distance (usually 4-5 m) as the material moves up
and down against the knife.
Cryostat
A microtome in a refrigerated unit. The cutting temperature is

commonly -20oC.
Blocks
This word is used interchangeably with pieces of tissue used in

histological analysis and cassette They are usually fixed and
paraffin embedded. The blocks themselves usually include a plastic
cassette containing the specimen number. The pieces may vary
greatly in size from tiny diagnostic biopsies (a few millimetres
across) to up to 2m in length. Larger pieces may be placed in mega
cassettes or mega block.
Mega blocks
Larger plastic cassettes containing tissue. They are big enough to

contain a full cross section of bowel or a cross section of an
enlarged prostate.
Tissue processor
A machine which processes tissue. Tissues are moved through a

variety of chemical reagents to dehydrate them and make them
suitable for placing in the embedding media, which is usually wax.
Embedding
media
The material which tissue is held within for cutting sections.

Embedding media may be wax or may be resin. The selection of
the embedding media is dependent on the hardness of the tissues,
harder tissues need a harder embedding media , and the thinner
the sections need to be cut then the harder the media needs to be.
Lesion
An area of suspected pathology, that may be well or ill defined,

pigmented or not, raised, smooth or textured.
SPECIMEN RECEPTION
It is essential that the material for investigation is received in the laboratory in a suitable
condition for testing. For the laboratory to handle specimens correctly laboratories need
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quite a lot of information about what the sample is, which patient it is from, who is
requesting the test and who wants the report.
THE SPECIMEN REQUEST FORM SHOULD CONTAIN INFORMATION ABOUT WHO M THE
RESULTS ARE TO BE SENT TO
The minimum specimen information should be
Full surname
Full forename
One other identifier e.g. Date of birth, NHS number, Hospital number.
What the sample is
The request form may also ask for all of these items (if appropriate) in addition to the
minimum data. The laboratory will provide instructions to users on how the specimen
should be taken and transferred to the laboratory. Figure 3.3 shows a typical specimen
reception area in a busy histology laboratory.
WHEN SPECIMENS ARE RECEIVED INTO THE LABORATORY IT IS IMPORTANT TO CHECK

THE DETAILS ON THE REQUEST FORM AND THE SPECIMEN POT. IF THERE ARE ANY
DISCREPANCIES FOR EXAMPLE IN THE NAME AND DATE OF BIRTH OF THE PATIENT ON
THE POT AND ON THE REQUEST FORM THEN THE SPECIMEN SHOULD BE RETURNED
TO THE PERSON REQUESTING THE SPECIMEN FOR CORRECTION. A RECORD OF THIS
ERROR PATTERNS/LOGS SHOULD BE MADE SO THAT ANY PATTERNS OF MISTAKE CAN
BE FOLLOWED UP.
What patient information is required to accept a specimen in a histology

laboratory?
CLINICAL INFORMATION
When samples arrive in the laboratory, the type of specimen and what the clinical history
is dictates the way samples are handled. If some vital clinical information is not supplied
then it is possible that diagnosis can be compromised, incomplete or incorrect.
THE CLINICAL HISTORY MATTERS. GOOD CLINICAL HISTORY HELPS GET A GOOD
REPORT
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For example liver biopsies can be handled completely differently based on the clinical
information provided. If patient A has had a previous colonic cancer and had lymph
node involvement with tumour presents with decreased liver function tests.
Immunocytochemistry will be required to investigate the presence of metastatic
cancer as this is the most likely differential diagnosis.. Patient B who had a history of
alcoholism and presents with decreasing liver function tests. Special stains will be
required to investigate primary liver function. This is summarised below.
Figure 1 - Figure showing the most likely lab tests required based upon the clinical details
provided for a liver biopsy
SUBSEQUENT TESTS WITHIN THE HISTOLOGY LABORATORY FOLLOW DEPENDING ON

THE INFORMATION SEEN ON THE FIRST SLIDES VIEWED
CLINICAL CORRELATION
The liver has many differed functions and a liver biopsy is a very valuable diagnostic tool
for assessing a range of disease states. In the histology laboratory about 6 extra stains
are performed. This gives lots of extra information
Full clinical information helps the laboratory to make a full diagnosis
A single patient may receive many biopsies. Therefore, it is important for the reporting
pathologist to be aware of the previous clinical history. Often a small diagnostic biopsy sometimes an endoscopic sample 2mm in diameter, or a needle core of tissue 2mm wide
by 10mm long, will be taken to assess the nature of the patient cancer. This diagnostic
sample will lead to a diagnosis and the planning of the patient treatment. The treatment
plan itself may result in further surgical procedures, known as a therapeutic biopsy or
resection.
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The storing of patient results enables any new pathology to be correlated with that of
previous samples. This is of particular importance when monitoring patients with
recurrent cancers.
ENDOSCOPIC BIOPSIES ARE BIOPSIES OBTAINED BY USING A FLEXIBLE TUBE

(ENDOSCOPE) WITH A GRIP FUNCTION ON THE END. THE ENDOSCOPE IS INSERTED
INTO THE MOUTH OR ANUS AND ALLOWS SAMPLES TO BE TAKEN FROM THE INSIDE
OF THE GI TRACT WITHOUT THE NEED FOR INVASIVE SURGERY
TIMELINESS
The processing of samples, and return of results, can be affected by a number of time
factors. For example, some laboratories can serve multiple hospitals, such that the sheer
workload may place a limit on how quickly a sample can be processed. Also, while most
laboratories provide a collection service for samples from GP surgeries within their
service provider areas, the size of collection areas may mean that collections may only
happen daily at best.
Most histology samples will not be available for reporting until the next working day from
receipt at the earliest. Any specimen requiring more urgent handling will need to be
highlighted and discussed with the laboratory.
CLINICAL CORRELATION
Patients with suspected cancer in the UK can expect to receive their results back within
14 days of the first GP appointment. Included in this time is the hospital appointment and
the laboratory diagnosis. Rapid results means the patients can be treated quickly which
helps with their clinical management.
Histology takes time

Results are only available same day when specially requested
URGENT SAMPLES
Urgent samples are received where the patient needs urgent clinical intervention based
upon the histology result. Samples may be urgent because a result is needed while the
patient is on the operating table, as in the case of patients requiring rapid frozen
sections, or they may have a rapidly declining medical condition which needs clinical
intervention. Patients who present with suspected cancer require their results back within
2 weeks so treatment can be planned.
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Urgent samples fall into two groups

1 Frozen sections
2 Urgent fixed specimens
FROZEN SECTIONS
Frozen sections are a way of reporting histology specimens rapidly. The specimens are
frozen to make them hard and then thin sections are cut, hence the name.
URGENT FIXED SAMPLES

Hospitals deal with many patients. Some can be treated in a measured manner with
planned surgery and treatment. Other patients present with symptoms only when they
are very ill. Patients who present with late symptoms are often difficult to diagnose and
therefore urgent histology samples are removed from the patient. They may be
endoscopic biopsies or needle core specimens. Urgent fixed samples are lesions or
nodules which can be excised as a discrete entity but clinicians are anxious to plan future
surgery or treatment.
A 63 year old male patient presents at his GP with a slightly itchy darkly pigmented
lesion with an irregular border. In recent months the lesion has grown significantly. See
fig 3.8 The GP suspects a malignant melanoma. A rapid appointment is made at the local
hospital dermatology department and the patient is biopsied 3 days later. The excised
skin lesion is placed in formalin and sent to the lab for urgent reporting. After processing,
3 levels are cut on the 3 blocks and the reporting pathologist suspects malignant
melanoma and immunocytochemistry is performed to confirm this. The case is reported
and the results sent back to the consultant dermatologist and the GP. The who process
from biopsy to report took 8 days, hence the process is urgent.
SPECIMENS NEED A REQUEST FORM WITH INFORMATION ABOUT THE PATIENT AND
THE TISSUE SAMPLE ON IT
What clinical circumstances might indicate an urgent specimen?
COMPETENCY ASSESSMENTS
SPECIMEN RECEPTION
You will be able to:
explain the purpose of the procedure
accurately maintain records
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demonstrate completion of procedure
Mostly
needed
prompting
Satisfactory
Good
1. State the purpose of the specimen reception procedure
2. State the health and safety risks of formalin
3. State the use of Michels transport media
4. Describe the specimen acceptance / rejection policy
5. Describe how specimens are allocated to consultants
6. Describe the purpose and limitations of frozen section diagnosis of histology

specimens
7. Why are specimens sometimes sent to referral centres
8. What constitutes a high risk sample? How should they be handled?
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9. Why are specimens from patients with a similar sounding name kept separate?
10. Why are specimens of the same type kept separate?
11. Why are gastro-intestinal specimens labelled in anatomical order?
12. Why are records of rejected specimens kept?
Task
Can clear up a small formalin
spill
Can clear up a large formalin
spills
Can use the respirator
Can explain / demonstrate how
to date and time stamp
specimens
Demonstrate the procedure for
dealing with empty specimen
pots
Demonstrate how specimens are
labelled up
Demonstrate how private
specimens are divided up
amongst consultants
labelled up
Can describe how red cross
samples are handled
prioritised for frozen sections
sent to referral centres
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Date
Assesse
d
Asses
sor
Assessors
signature
Candidates
Signature
TELEPATH
Task
Date Assessed
Assessor
Assessors
signature
July 18,
2011
Candidates
Signature
Can book a
specimen in on
telepath using
RXR number
match
Can HPROE a
specimen to
ensure block
and slide
details match
IBMS PORTFOLIO CROSS-REFERENCE

2a.1, 2a.2, 2a.3
SPECIMEN DISSECTION ROOM

FIXATION
A majority of routine specimens received in the laboratory are suitable for
formalin fixation, but not all of the subsequent tests required are compatible with
fixation. Therefore before the sample is taken it is important to know what tests
will be required and arrangements made to receive the samples either fresh or in
Michels transport media in the case of skin samples for
immunofluorescence. When cells are in the body they receive oxygen, food
and have waste products removed. When they are removed from the body they
decline and die; due to the absence of oxygen, absence of food and the build up
of waste. Cells start to change, the DNA, proteins, sugars and fats all start to
deteriorate initially through the build up of waste products and the uncontrolled
release of proteases. This process is known as autolysis. Autolysed cells stained
with haematoxylin and eosin show enhanced cytoplasmic staining or
eosinophilia (i.e. more pink) and a reduced nuclei staining (less purple) this is
due to the breakdown of RNA molecules in the cytoplasm and the breakdown of
DNA molecules in the nuclei. Later bacterial or fungal contamination can cause
further destruction of cells and tissues. This process is known as putrefaction. If
the shape and structure of these cells are allowed to deteriorate then diagnostic
analysis becomes impossible. The first part of the histology process is therefore
making sure the cells do not deteriorate. Prevention of cellular deterioration is
done by one of two processes - fixing or freezing. The nature of preservation in
histology is to keep cells in as life-like state as possible. For example, onions are
pickled in vinegar to preserve them and keep them edible. At a cellular level the
process of autolysis is seen as karryorhexis and apoptosis. The freezing of
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histology samples occurs only in the minority of cases and will be due to the case
being required for tests which are incompatible with the chemicals used in
fixation. Examples of tissues which should be frozen on receipt are muscle
biopsies, specimens for immunofluorescence and suspected rare tumours were
molecular studies may be of interest.
Autolysis is the process of cellular degradation by the release of intracellular enzymes

Eosinophilia is enhanced staining with eosin, sections will look much more pink than
usual
Michels transport media is a buffered aqueous salt solution used for transferring
biopsies for immunofluorescence to the laboratory
Putrefaction is decomposition of cells by bacterial or fungi.
Karryorhexis - is the destructive fragmentation of the nucleus of a dying cell whereby its
chromatin is distributed irregularly throughout the cytoplasm.
Apoptosis is the process of programmed cell death. The nuclei is seen to reduce in size
and stain much more intensely with attachment, cell
shrinkage, nuclear fragmentation, chromatin condensation, and
chromosomal DNA fragmentation.
Pyknosis Fixation - is the process of chemically preserving cells
Admiral Nelson was placed in a barrel of rum after being shot dead on HMS
Victory. The artist Damian Hurst has placed sheep and sharks in formalin to
preserve them. In histology formalin is also used as the main preservative of
cells.
Fixation aims to preserve cells and tissue constituents in as close to a life-like state as
possible. Essentially it is concerned with the stabilization of proteins within the tissue and
arresting the effects of autolysis and bacterial decomposition. However, cell preservation
is only part of the fixation effect. Fixatives impart additional benefits for the histologist
as they allow the tissues to undergo further preparative procedures without change and
make the cellular components more easily colourable by dyes. It is important to realise
that the appearance of the tissue after fixation is artefactual. The stabilization of proteins
is necessary to prevent their diffusion during subsequent processing but this also
changes the structure and appearance of these proteins.
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Fixation aims to preserve cells and tissue constituents in as close to a life-like state as
possible
Information is available when the nucleus is examined. The size shape, texture and
staining properties of the nucleus alter in differing pathological conditions. .
Not all specimens received in the laboratory are suitable for fixation. This is because the
subsequent tests required on some specimens are not compatible with fixation. Before
the sample is taken it is important to know what tests will be required. Having said this,
the majority of specimens received in the lab are received in fixative which is ideal for
the vast majority of routine samples. The others are most probably received either fresh
or in Michels transport media. Michels transport media is not a fixative but a solution
of salts plus an antibacterial agent, which acts to sustain biopsies in an osmotically
balanced solution until transported to the laboratory for treatment can be performed.
Clinical correlation
Much of the key clinical information is linked to the size and
shape and staining properties of nuclei. Good fixation is critical
for good preservation of nuclei.
PRINCIPLES OF FIXATION
There are a number of features that make a fixative ideal. They are:Table 3.1 - Features of an ideal fixative
Factor
Reason
Kill cells quickly and evenly
This stops the build up of harmful

metabolites which disrupt the cellular
appearance
Penetrates tissues quickly and evenly
The solution diffuses through the tissue

quickly
Prevents autolysis and putrefaction
Stops cellular functions immediately

preventing self destruction and
prevents microbial damage
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Factor
Reason
Does not shrink or swell the cell
Maintains the normal shape and size of

the cell in relation to the size in situ.
This allows for meaningful diagnostic
and prognostic measurements.
Prepares the tissue for later treatments
Allows the use of the tissue for a large

number of further tests. This includes
making the tissue harder
Prevent desiccation and drying of tissue
Keeps the cells hydrated
Safe to use
Does not cause harm to the operator

when using the fixative. Does not
require expensive disposal regimes.
Reasonably priced
As large quantities of fixative are used

in the routine laboratory. The cost
should not be prohibitive
Tolerant in use
Specimens are likely to be placed into

fixative by non histology staff in many
different ways. A tolerant fixative allows
various permutations of usage not to
prevent
NO FIXATIVE IS IDEAL FOR ALL CIRCUMSTANCES
Often salt or buffer is included. The salts are added to adjust the osmolarity of the
solution. Ideally the fixative solution should have the same salt levels as the cells being
fixed. This ensures that cells neither shrink nor swell. Figure 3.6 shows how cells alter in
solutions of differing tonicity.
MECHANISMS OF FIXATION
Fixation is a chemical process where the constituent molecules within the cells can be
manipulated in several ways:-
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Cross linking (or additive) fixatives act by binding amino acids within proteins to
adjust their 3D structure, preventing autolysis and putrefaction. Formalin fixation is not
suitable when handling specimens for enzyme histochemistry since the cross linking acts
to denature many enzymes.
Precipitating or (non additive) fixatives act by removing water from the cellular
matrix, this acts to disrupt the 3D (tertiary) protein structure and therefore, precipitating
the protein..
Table 2 Table showing the various types of fixative in common use and their mode of action.
Cross linking
Precipitating
Compound
Glutaraldehyde
Alcohol
Picric acid
Formaldehyde
Acetone
<KP> The aim of fixation is to prevent the decomposition of cells by intracellular

processes and external action of bacteria, preserving cells in a balanced isotonic solution.
The use of fixation reagents results in significant changes to the tissue structures and
components. These changes are beneficial in terms of tissue preservation and arresting
the decomposition effects described previously. However it is important to recognise that
some tissue components are extremely labile and so would be readily inactivated by the
use of these chemicals. As such it is important to know what investigations may be
required for individual samples so that the appropriate processing regimes can be
employed. Fixation is a reversible process and much effort is taken to undo the effects of
fixation e.g. in immunocytochemistry, during antigen retrieval, you will read more
about this in chapter 5.
Labile able to under go change easily i.e. unstable
Well fixed cells have good nuclear detail they are neither swollen nor shrunk, the
integrity of the general architecture is not disturbed and cellular proteins are well
preserved for future tests.
Poorly fixed cells have poor nuclear detail; they may be swollen or shrunk. The general
architecture of the piece of tissue may be distorted and cellular proteins may be lost.
Tissue which is fatty is very vulnerable to poor fixation as the aqueous reagents do not
penetrate the tissue well.
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POOR FIXATION CAN LIMIT THE POSSIBILITY OF A DEFINITIVE DIAGNOSIS
Tissue fixation is usually achieved by means of chemicals but the same or similar effects
can be achieved by the use of heat. Heat is used to precipitate proteins and to prevent
degradation. When heating care must be taken not to overheat or damage the tissue.
Microwave ovens can be controlled to heat at a steady temperature, neither too hot nor
too cold. Heating also allows fixation to take place at a much faster rate, reducing the
fixation time from hours to minutes. Heat can also be used in combination with chemical
fixatives. More of this later in the chapter when we talk about automated processors.
FACTORS AFFECTING FIXATION

Fixation is a chemical process and is affected by environmental conditions.
Table 3.3 Factors affecting the rate of fixation

Temperature
The hotter the fixative media the quicker the fixation. Microwave
processors utilise the heating of fixatives to allow quick processing
schedules.
Size of
specimens and
penetration of
fixative
Large specimens take longer to fix than small specimens. The

penetration rate is the rate at which the chemicals permeate the
tissue. This may be only a four or five millimetres a day,
consequently large resections need slicing open before the
processes of autolysis destroys the detail within the tissue.
Changes of
volume
The larger the volume to specimen the more rapidly fixation will
take place. It is recommended that there is 20x as much fixative as
specimen. Often this is not possible, due to the resection being
large.
pH and buffers
The addition of buffers does slow the fixation process slightly, yet it
does ensure that the chemicals are at the correct pH. Acidic
formalin fixatives react with tissues and can create pigments.
Osmolarity
Fixatives should be the same osmolarity as the cells they are fixing.
This will help to prevent shrinking or swelling.
Concentration of
fixatives
The stronger the fixative the more rapidly fixation takes place i.e.
the more active ingredients for the fixation reaction the more
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rapidly this can take place.

Duration of
fixatives
The longer tissues are in fixative the more complete the fixation will
be.
Table 3.4 near here
KEY FIXATIVES
There is not a single fixative that is suitable for all preparations. Many different fixatives
exist although the number in routine use is now relatively small.
The most commonly used fixative in the UK routine laboratory is formalin.
It is important that laboratories use similar fixation regimes as this allows laboratories to
review each others cases more easily and to compare methodologies. Bouins solution is
occasionally used for testicular biopsies due to enhanced nuclear detail.
FORMALIN
Formalin is cheap, easy to make, keeps well on the shelf and is easily transported.
Formalin is a solution of formaldehyde gas contained within water. Formalin is an
aqueous acidic solution that contains a concentration of formaldehyde gas dissolved
within it. Acid formalin causes formalin pigment, a brown residue seen in red blood
cells.. When the pH is neutralised formalin pigment is not seen, hence in most
circumstances buffering agents are added. When purchased commercially within the UK
it is usually supplied at a concentration of 37-40% formaldehyde. Traditionally this
solution has been considered to be 100% formalin. This solution is usually diluted 1/10 to
give a formaldehyde concentration of 4%. This 10% formalin solution is the most
commonly used fixative in routine laboratories.
Formalin has a number of health and safety considerations which impinge on the running
of the laboratory.
The fixation reaction is reversible and is exploited in immunocytochemistry in antigen

retrieval.
Methylene bridges are -CH2- groups which link proteins together
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Health and safety - Formalin

Colourless liquid
Causes burns
Very toxic by inhalation, ingestion and through skin absorption
Readily absorbed through the skin
Possible human carcinogen
May cause allergic reactions
Causes watery eyes at over 20ppm
NON FORMALIN FIXATIVES (PRACTICAL ASPECTS)

Despite its tolerance and general all round suitability, formalin is not the best fixative in
all situations. Exceptions to the use of formalin as the initial choice of fixative are:
Specimens requiring electron microscopy here the use of glutaraldehyde (despite its
inherent health& safety issues) is still the agent of choice.
GLUTARALDEHYDE
Glutaraldehyde provides excellent ultrastructural preservation and is the fixative of
choice for electron microscopy. It fixes tissue rapidly but does not penetrate tissue deeply
making it unsuitable for large samples. Specimens for electron microscopy are often cut
up into 2mm cubes which allows for fixation throughout the whole tissue. Glutaraldehyde
is a respiratory sensitiser and needs to be handled with care.
ULTRASTRUCTURE REFERS TO THE PRESERVATION OF THE ORGANELLES WITHIN

THE CELL I.E. MITOCHONDRIA, GOLGI COMPLEX
RESPIRATORY SENSITISER IS A CHEMICAL WHICH IRRITATES THE LUNGS AND MAY

CAUSE ASTHMA
BOUIN'S
Bouin's solution is a mixture of picric acid, alcohol, formaldehyde. This yellow compound
fixative is popular for the fixation of testicular biopsies, due to the improved nuclear
chromatin appearance of the spermatogenic cells produced with this solution.
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Others used even more rarely include Zenkers fixative, Industrial methylated spirits,
methanol, acetone, formaldehyde vapour and commercial preparations.
MICROWAVE FIXATION
The stabilization of proteins can also be achieved by the use of microwave irradiation.
This has the advantages of significantly improving the speed at which fixation can be
completed and the absence of any noxious chemical fixing chemicals.
An alternative approach is to supplement the usual chemical fixative with microwaves to
speed up the fixation process. There is currently great interest in developing microwave
based automated tissue fixation and processing machines.
SPECIMENS NOT FOR FIXATION

Not all specimens to be investigated require fixation. Some specimens need to be
handled more quickly than fixation can take place i.e. in urgent cases. Some tissue
elements react with the fixatives in a way that is unsuitable for diagnosis, such as
immunofluorescent samples and some cells are required to be kept alive i.e. cytogenetic
samples.
CYTOGENETICS
Cytogenetics is the study of the structure of chromosomal abnormalities by analysing
chromosome numbers. Tissue samples for cytogenetic analysis must be transferred in
cytogenetic culture media directly to the cytogenetics laboratory.
Cytogenetics is a branch of laboratory science which looks at chromosome numbers.

Cytogenetics is of particular interest in fetal and perinatal samples. A count of the
chromosomes can aid the pathology diagnosis and explain the clinical presentation. In
the cytogenetics laboratory tissue samples are separated into cells and cultured to
propagate them - the resultant cell harvest has its DNA extracted.
HOW DO I KNOW MY TISSUE IS WELL FIXED?

The quality of fixation within tissue is seen usually through two aspects
Morphology the shape of the cell, their constituents and the tissues
Staining quality the specificity of the staining seen
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Red blood cells should look bi-concave.

Chromatin within the nuclei is crisp and shows aggregation into clumps towards the cell
membrane.
DECALCIFICATION
After tissues have been fixed they are then ready for the next stage of laboratory
investigation. Some tissues cut easily with a knife and require no further pre-treatment.
Some tissue structures e.g. bone and teeth are extremely hard due to the presence of a
calcium phosphate salt (hydroxyapatite). This mineral is essential in life for these
structures to perform their role but for the histologist unless this mineral is removed then
the production of thin paraffin sections for microscopy will be problematic. The method of
achieving this is termed decalcification as it is primarily calcium salts which are removed.
However, during the process materials other than calcium will also be removed so a more
accurate term to apply would be demineralization.
Calcium crystals may also be present in other tissues as part of a pathological process.
Tissues such as breast may be x-rayed prior to being processed) . The identification of
this calcium has diagnostic significance and therefore it is important that the calcium
remains in the tissue.
DECALCIFICATION IS ESSENTIAL FOR THE SECTIONING OF PARAFFIN PROCESSED

TISSUE
There are certain bony samples which are not suitable for decalcification, they are
samples where disturbances to calcium metabolism is suspected e.g.Osteomalacia
(adults) or Rickets (children). In these conditions the amount of mineralized to nonmineralized bone is reduced. In order to be able to diagnose these conditions it is
necessary to visualize the extent of both and if the specimen is decalcified then it will
impossible to make this estimation. In these cases the sample must be treated by
embedding the still mineralized bone into a hard embedding medium such as a plastic
resin. This will permit suitable quality sections to be produced and stained.
Whichever decalcification method is employed there is a balance to be struck between

speed of decalcification and preservation of cellular morphology. Generally the speedier
the rate of decalcification the greater will be the rate of destruction (maceration) of
cellular morphology. This effect is exaggerated if tissue fixation has not been completed
satisfactorily prior to commencing decalcification. The choice of decalcifying method and
chemical will be heavily influenced by whatever the sample is being decalcified for i.e. a
patient's bone sample being decalcified for a possible clinical diagnosis of malignancy
Page 20 of 61
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will be required much more rapidly than a similar sample being treated for training and
education purposes only. The strength of the acid affects the speed of decalcification.
Factors affecting the rate of decalcification are:Concentration of the acid within the decalcifying agent
Level of agitation of the tissue
Saturation of calcium ions within the decalcifying solution
MACERATION THE LOSS OF NUCLEAR AND CYTOPLASMIC DETAIL THROUGH

AUTOLYSIS.
EXCESSIVE DECALCIFICATION OF TISSUES IN ACID CAN CAUSE TISSUE DAMAGE OR
MACERATION.
DECALCIFYING AGENTS
Decalcifying agents fall in to two main categories: acid decalcifiers and chelating agents.
Acid decalcifying agents act by reacting with the calcium in the tissue to form soluble
salts.
Nitric acid very fast acting but produces the most tissue damage. It is suitable for the
controlled decalcification of very hard bones such as skull.
Hydrochloric acid can be used alone or frequently as an ingredient of commercially

available decalcifying solutions. Fast acting but produces less tissue damage than nitric
acid.
Formic acid much slower than nitric or hydrochloric acids and so is gentler to the
tissue. It is probably the most popular choice of decalcification fluid for most diagnostic
histopathology applications.
Commercial solutions a number are available within the UK market. As mentioned

above most use hydrochloric acid but some use formic acid. Recently recipes containing
formic acid and formalin have been promoted which are claimed to permit 'concurrent
fixation and decalcification' which contradicts the traditional edict mentioned previously.
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Chelating agents offer alternatives to the use of acids. They act as 'calcium sponges'
continuously mopping up the free ionic calcium that is always present around the
mineralized bone. Chelating agents offer a more gentle approach and may be the
decalcifying agent of choice in tissue which is likely to require immunocytochemistry.
Ethlenediaminetetraacetic acid (EDTA) this is the main chelating agent used.

Calcium removal is very slow using this method and as a consequence there is minimal
tissue damage. Probably the best option if there is no pressing demand for a speedy
result. EDTA is the decalcifying agent of choice if immunocytochemistry is likely on the
specimen.
Trisodium citrate has a similar rate of demineralization as with EDTA.
SPECIMEN DISSECTION
The dissection of the specimen is carried out by trained biomedical scientists or
pathologists depending on the specimen complexity. Each specimen is categorised A
through to E for the complexity of the dissection (see table 1 for an explanation of the
specimen categorisation groups). Sampling requires the tissue to be examined
macroscopically (i.e. by eye), and only elements that appear to be of clinical significance
are selected and submitted for histological examination. A robust knowledge of anatomy
and pathology is required to select the correct tissue samples.
The Royal College of Pathologists lays out the minimum assessment criteria to be
reported on for most major cancer types e.g. bowel, breast, skin, liver, prostate and
kidney. The assessment criteria are known as minimum data sets and each malignant
sample should have each criteria of the minimum data set included in the final report.
When specimens are dissected it is important that the selection of tissue leads to two
points, firstly diagnosis of the disease and secondly the proximity of the disease to the
margin, the depth of any invasion into the tissue and the involvement of any lymph
nodes. Reporting on these criteria allows clinicians information on which to base patient
management and decide whether they need further treatment or not.
Key point
Cellular pathology reports on cancer cases are required
to contain key information relating to the tumour type,
involvement of lymph nodes with cancer and presence of
metastases
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<KT> Minimum data set A set of information that each malignant case reported should
contain. The information required includes size and grade of the tumour seen, distance
from resection margins and presence of metastases in the lymph nodes or distant sites.
Table 3.4 - Table showing the various dissection categories of specimens with examples
Specimen
category
Process
Examples
Simple transfer of biopsies into a processing

cassette. No dissection of the tissue is required
Gastric biopsies,
Specimens which require routine simple

dissection. Usually no more than 2 3 blocks
Gall bladder, Appendix
Specimens which require a standard dissection

i.e. set samples form set locations
Uterus and cervix
Specimens which require interpretive sampling

of the specimen
Colon
Complex anatomical specimens which require

extensive sampling
Neck dissections,
Whipples procedures
Cystectomy
Vulvectomy
Whatever is being dissected it is important to realise that the selection of samples

determines more than any other step the suitability and quality of the finished report.
Case study
A 51 year old lady presents at clinic with a firm breast lump,
after an initial mammograph and core biopsy a ductal carcinoma
is confirmed. A subsequent breast lump is excised. The
specimen is inked intact so that the edge of the tissue or margin
can be identified at a later date. Once the outer features have
been described (i.e. what is it? How big is it? Is there skin
attached? Is there a lesion easily identified?), then the specimen
is sliced and any features are noted
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Good selection of tissues for processing is also important, sometimes standard cassettes
are not sufficient to allow for a whole cross section of tissue to be examined in one piece
and therefore larger pieces of tissue are required to be sampled in a single piece i.e.
colon specimens and specimens of larynx to be placed in their entirety in a single block.
This can be achieved by loose processing of tissue or the use of Mega or Jumbo
cassettes, which measure 6 x 5x 1.5cm. The advantage of this is it allows for much easier
anatomical orientation of tissue.
Key point
Cassettes that are overfull can present problems for processing
embedding and sectioning
Additional cassettes may seem to be adding to the total workload but in all probability
the subsequent embedding and microtomy will be much easier and so too will the quality
of the end result. It is important to ensure that all stitches and staples are removed prior
to processing as they can cause great difficulty when attempting to section blocks. The
lids are added to the cassettes and they are ready for the next stage of the process.
<KP> Each tissue block selected at specimen dissection should have an unique identifier
on it stating the case number, the specimen pot identifier and the block number. This
allows for unique identification of each block.
Health and safety

It is essential that the specimen dissection area is cleaned of any
bits of specimen after each case. Cross contamination of
material can cause misdiagnosis!
<KP> The macroscopic specimen report should be sufficiently clear so that anyone
reading the report can identify what features have been identified and where tissue
blocks have been taken from
SPECIMEN STORAGE AND DISPOSAL

After dissection any remaining tissue is kept in an air extracted storage facility. The
specimen is kept for a defined period of time, usually around 4 weeks after the sign out
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of the specimen report. The storing of specimens in this way allows subsequent
investigations on the tissue for one of a number of reasons
Initial pathological lesion not identified
Unexpected findings on the initial examination microscopically
Further clinical information becoming available
COMPETENCY ASSESSMENT
ASSISTING WITH CUT UP
Level
1
1
1
1
1
2
Competence
Assessed
competent
Assessor
Countersigned by
candidate
Can perform
patient
demographic
check
Can describe
and process
an endoscopic
biopsy
Writes number
of pieces on
casette side
Can set up for
cut up
Can
demonstrate
daily clean up
Can
demostrate
end of week
clean up
Read SOP
Can describe
and process all
other
specimen
types as per
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Level
2
2
BMS CUT UP
Page 26 of 61
Competence
SOP CP/H2
Completes
competency
assessment
CP/H2C
Can take a
dictation of
specimen at
cut-up
accurately
using
appropriate
abbreviations
Can mark
cassettes for
ribbons, levels,
stains and
orientation
Can
demonstrate
what to do is
specimens
need further
fixation
Can accurately
record details
on request
form
Can trouble
shoot
discrepancies
Can prepare
racks for
processing
Assessed
competent
Assessor
Countersigned by
candidate
Level
Competence
Read SOP
1
2
July 18,
2011
Assessed
competent
Assesor
Countersigned by
candidate
Assessed
competent
Assesor
Countersigned by
candidate
Can perform
patient
demographic
check
Can describe
and process
an endoscopic
biopsy
Writes number
of pieces on
casette side
Can describe
and process all
other
specimen
types as per
SOP CP/H3
Completes
competency
assessment
CP/H3C
DECALCIFICATION
MACROPATH USE
Level
Competence
Has read SOP
1
1
Can prepare
10% formic
acid
Can prepare
block for decal
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Can maintain
decal log book
Can change
decal reagent
Completed
competency
questions
Can
demonstrate
end point
decalcification
testing
2
2
X-RAY USE
Leve
l
Competence
Read SOP
Can switch on and

prepare x-ray machine
Can prepare specimen for

x-ray
Can load specimen into xray machine
Can select the

appropriate time and KV
1
1
Can perform X-ray

Can safely remove
speciem and x-ray
casette
Can go to radiology to
develop x-ray film (BGH
only)
Can operate safely
Can switch on and
prepare x-ray machine,
Page 28 of 61
Assessed
competent
Assesor
Countersigned by
candidate
Leve
l
Competence
Assessed
competent
July 18,
2011
Assesor
Countersigned by
candidate
Assesor
Countersigned by
candidate
PC and start faxitron

software
Can enter patient details
to start new case
Can perform x-ray of
small specimen using
faxitron software
Can perform x-ray of
large specimen using
large x-ray plates
Completed competency
questions
SPECIMEN STORAGE AND DISPOSAL
Level
Competence
Can select
specimens from
the shelf for
discard
Can discard of
formalin and place
specimens in
clinical waste bag
Can tag up
specimens for
anatomical waste
Can remove
waste from the lab
Assessed
competent
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2011
Level
Competence
Assessed
competent
Assesor
Countersigned by
candidate
Can discard small

specimen pots
Can clean buckets
TISSUE PROCESSING AND EMBEDDING

TISSUE PROCESSING
The aim of tissue processing is to prepare tissue in a supporting media ready to be cut in
to thin sections, so that very thin slices of the tissue can be cut in order to observe
individual cells under the microscope. This means being able to produce slices (sections)
at a thickness of between 2 and 6m, about the thickness of a single cell or 1/10th the
thickness of a human hair. The most commonly used embedding media is paraffin wax.
When solid, paraffin wax has a consistency very similar to that of the tissue itself and this
assists with the subsequent sectioning process. Wax however is immiscible with water so
the tissue can not simply be transferred from water to wax. Instead, the tissue must
transfer through a number of intermediary steps until it is able to be impregnated with
wax. This is termed paraffin processing.
The tissue blocks prepared during specimen dissection are immersed in a number of
different chemical solutions. These solutions act to prepare the tissue for mixing with an
embedding media, in most cases paraffin wax although resin is sometimes used for hard
tissue and material which needs to be cut into sections thinner than about 2 m (about
half the normal thickness). Paraffin wax is the embedding media of choice because it is
easily handled, molten and solid, is non toxic, and is hard enough to support the cutting
of thin sections.
Key point
Paraffin wax is the embedding media of choice because it is
easily handled, molten and solid, and is hard enough to support
the cutting of thin sections.
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Histology specimens are generally run in batches overnight although continuous

throughput or Lean Technology is now being applied so that smaller batches can be
processed, so that peaks and troughs of workload can be eliminated. This results in the
service being more efficient and reduces result turnaround time for the results of some
samples. Additional processing schedules exist for small urgent pieces of tissue or for
larger pieces which require longer time in the reagents.
Processing is not always straight forward. Different types of tissue require slightly
different processing regimes. In most laboratories this is not always possible as there will
be a range of different tissue types appearing on any one day, a limited amount of tissue
processors and limited time. Generally processing is a compromise of a number of
different factors to give the best overall balance of quality of processing, timeliness and
cost. Tissues are generally processed together in an overnight batch, but this may result
in poor processing of fatty tissue and particularly hard tissue.
Lean technology is the concept of derived from manufacturing

industry. It is the process of analysing the steps of the process to
ensure the most efficient work patterns. Within tissue processing
this is the concept of reducing batch size to the smallest number
possible with the aim of ensuring work moves progressively
through the laboratory without any waiting steps.
Key points
The size of the tissue dictates how quickly a block may be
processed, small tissue pieces can be processed quicker than
larger pieces
The embedding media should be the same hardness as the tissues being cut. If the tissue
is particularly soft then a softer wax will be used. Brain is a particularly soft tissue, with a
consistency of solid custard and therefore, neuropathology laboratories commonly use a
mixture of paraffin wax and dental wax (a much softer wax dentists use to get patients to
bite in to when taking a dental impressions) in their infiltrating stage, this will give the
wax a pink colour but also makes it more appropriate for sectioning brain tissue.
For tissue processing it is convenient to consider tissue processing in 3 distinct stages:
Table 3.5 - Table showing the three stages of tissue processing
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Dehydrati
on
Removes water from the section,
Clearing
to act as a link between the dehydrant and wax. The solution must be
miscible with both the dehydrating agent and the wax. Clearing is the
term that was applied due to the observed effect that some of these
chemicals had upon the tissues. Many have a similar refractive index to
the tissues and this caused a resultant transparency or 'clearing' of the
tissues after immersion for a suitable time. Not all of the reagents used for
this purpose actually clear the tissue so other terms have been suggested
e.g. ante-media but the use of clearing agents has remained steadfastly
popular. Most of these chemicals are organic solvents and all are toxic to a
greater or lesser degree. The purpose of clearing agents is to act as a
reagent which is miscible with both alcohol and wax. Xylene is probably
the most widely used agent but it suffers from rendering the tissue more
brittle and harder than many of its alternatives, particularly if incubation is
protracted.
Impregnat
ion
to infuse all parts of the tissue structure with molten wax in preparation
for subsequent embedding into a solid block.
REAGENTS
Several containers of each reagent are used to reduce the effect of contamination from
previous reagents.
A number of dehydrants are available but the most popular are industrial methylated
spirit (IMS) which is primarily ethanol and iso-propyl alcohol (synonyms iso-propanol,
propan-2-ol and 2-propanol).
Some authors have described the use of iso-propyl alcohol as both dehydrant and
clearing agent see xylene free processing.
Whichever option is chosen it is preferable to start with a more dilute alcohol and
gradually increase the concentration rather than to immediately introduce a higher
alcohol concentration as the tissue is more adversely affected by the latter option by
displaying increased shrinkage artefacts.
Health and safety Industrial methylated spirit

Highly flammable
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Harmful by inhalation and if swallowed

Requires specialist disposal
Tissue processing is a reversible process and by placing wax blocks in the processing
reagents in reverse order allows tissue to be taken back to an aqueous state.
Health and safety xylene

Flammable.
Harmful by inhalation and in contact with skin.
Requires specialist disposal
Irritating to eyes and skin.
XYLENE-FREE PROCESSING
Interest in the use of processing regimes which do not require the use of xylene or similar
clearing agents is largely due to the health and safety concerns associated with the use
of these chemicals and a desire to eliminate their use in laboratory practice. Advances in
processor technology have reduced the effect of problems that were encountered with
this concept in previous years.
Iso-propyl alcohol (IPA) can be used either in combination with ethanol, where the IPA is
used in place of xylene, or as both dehydrant and clearing agent. In either situation the
ability to use high temperatures within the modern tissue processor retort improves the
rate at which IPA is driven from the tissue as molten wax is introduced.
PROCESSING FATTY TISSUE

Because of their high lipid content these types of specimens may need extended times in
processing reagents, particularly dehydrant and clearing agents in order to achieve the
required effect. This may mean that these types of specimen are segregated from others
and processed by a separate regime created specifically for this purpose. In addition, if
xylene free processing is being considered then additional time may be required to
ensure complete removal of iso-propanol prior to wax impregnation.
HARD TISSUES
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Hard tissues such as tendon, nail, decalcified bony structures etc, and may benefit from
pre-treatment with tissue softening agents, either phenolic based solutions or surfactants
before final processing. This helps to soften the tissues for easier sectioning.
RESIN PROCESSING
Resin processing is an alternative to paraffin wax processing. Tissue is fixed and
dehydrated in the same way, instead of xylene a dilute resin mix is used and
progressively an increased amount of the resin is increased until the tissue is immersed
in pure resin. At this point the resin is cured and a hard supportive media surrounds the
tissue.
Key point
The harder the supporting embedding media, the thinner
the tissue can be cut.
TYPES OF PROCESSING EQUIPMENT

Activated charcoal filters are a constituent part of all enclosed tissue processors. The
purpose of the filters is to remove solvent and formalin vapours that are a by-product of
the pumping of reagents in and out of the reaction chamber. The activated charcoal
filters act to react with the solvent vapours released by the chemicals. The vapours are
locked into the filter and are rendered safe.
The quality of tissue processing is directly related to the

reagents used.
TYPES OF TISSUE PROCESSOR

How does tissue processing take place? Tissue processing may be performed manually,
with the histologist moving the material by hand or may be automated. There are a
number of different types of processor, carousel, enclosed process flow and microwave,
all of which work on the same principle i.e. bringing tissues into contact with processing
solutions for a fixed period of time then transfer to the next solutions. We will look at
each processing type in turn.
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Hand processing requires the tissues to be moved by hand between the reagents. This is
a very time consuming process and in practice, this does not happen often. Generally it is
only used for small urgent diagnostic biopsies. The reagents can be warmed to enhance
processing speed.
Factors affecting processing

Heat
Mixing
Vacuum
Wax Processing
Automated processing schedules are either carousel, enclosed, process-flow or

microwave. Most laboratories operate routine processing with enclosed processors but
process flow machines are becoming of increasing interest in large laboratories. The
major drawback with any automated processor is that the flexibility to adapt regimes for
different tissue types is lost as all specimens undergoing a particular protocol will all be
treated identically.
Carousel tissue processors originated in the 1950s and represented the first attempts
to automate tissue processing. In essence they copied the manual processing regime and
automated the transit from one processing reagent to the next using timer clocks. Their
use meant that many more samples could be processed at any one time and in particular
this automated transfer facility meant that processing schedules could be run overnight
and timer delays incorporated into processing cycles so that schedules could be operated
over weekends and public holidays. A major drawback with carousel processors is that
the tissue samples are extremely vulnerable whilst being transferred from one processing
reagent to the next. If an untoward event occurs during this process e.g. power failure
then the sample may be left out of fluid for some considerable time and the processing
solvents will rapidly evaporate from the tissue causing it to dry out and be unsuitable for
diagnosis. In addition when transferring from one reagent to another, there is
considerable release of flammable vapours with a carousel system, posing a considerable
fire risk. Many diagnostic samples are irreplaceable and so it is probably because of this
limitation more than any other that during the 1980s manufacturers began to develop
enclosed tissue processors.
Enclosed processors contain all the reagents within the machine. The tissue blocks are
contained within a chamber and the reagents are pumped in. Figure 3.17 shows a
diagram of an enclosed batch processor. The length of time each reagent is in contact
with the specimens is computer controlled. Enclosed processors have developed
Page 35 of 61
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increasingly complex safety systems to ensure the integrity of samples and to monitor
reagents. If a blockage prevents the entry of a solution the processor may skip to the
next reagent or incubate further with the previous reagent. Figure 3.18 shows a
photograph of an enclosed tissue processor
The process flow tissue processor allows material to be added to the processor at varying
points during the day. This way of handling tissues offers a Lean way of processing
material when it is needed, usually in more frequent, smaller batches. The principle of
the machine is very similar to the carousel processor where the specimen moves through
a series of reagents.
Microwave processing is a way of increasing the speed of tissue processing by using the
microwave process. Standard processing reagents of industrial methylated spirit and
xylene can not be used. Microwave processing technology is still in its infancy and some
processing machines require the manual changing of reagents.
Hand processing
Tissues can be processed manually. In practice this does not happen often and generally
it is only used for small urgent diagnostic biopsies. The reagents can be warmed to
enhance the processing speed.
Method hand processing
All reagents are warmed to 45oC before use
Formalin (15minutes)
70% alcohol (15 minutes)
Xylene (15 minutes)
Xylene (15 minutes)
Wax (15 minutes)
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Wax (15 minutes)
EMBEDDING
Once tissue has been processed it needs embedding in a manner to allow sectioning. The
aim of embedding is to orientate tissue to allow for the maximum amount of diagnostic
information to be retrieved. This involves ensuring the macroscopic cut surfaces are flat
on the base of the embedding mould and the tissue is correctly orientated. Most material
is embedded into a mould and has the plastic cassette which the tissue is placed in
attached to the back. This allows the material to be held in a manner which allows the
tissue to be gripped firmly in a clamp and thin sections to be made. Figure 3.20 shows a
photograph of embedding. The way in which tissue is placed for embedding is very
important. Correctly orientated tissue allows viewing and interpretation to happen easily.
Incorrectly orientated tissue prevents this. Large square samples from resections must be
placed flat within the tissue mould to allow for an even cross section of tissue to be
prepared
Method embedding
Vessels always need to be orientated in cross section e.g. Vasa
deferentia, Fallopian tubes, Temporal arteries
Skin sections need to be orientated so that the skin surface and
dermis can be seen in cross section.
Endoscopic biopsies should be embedded orientated.
Some colorectal biopsy samples.
THE ORIENTATION OF SPECIMENS AT EMBEDDING CAN HAVE A DIRECT
CONSEQUENCE ON THE ABILITY TO DIAGNOSE THE PATHOLOGY OF THE
SPECIMEN
EMBEDDING EQUIPMENT
For most laboratories, the volume of work requires the use of a designated embedding
centre (see figure 3.22 for a photo of the embedding centre). Most histological equipment
suppliers produce their own equipment centres but all have the same basic
requirements:
An electrically heated storage well to store the tissue cassettes waiting embedding and a
separate electrically heated storage well to store the base moulds.
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An electrically heated and thermostatically controlled storage tank for paraffin wax.
There is also an outlet nozzle from this tank which allows the operator to dispense the
required amount of molten wax as required.
An electrically heated 'warm' area which allows the operator to move the blocks around
the machine without everything sticking together.
An electrically controlled chilled area which allows the operator to orientate tissue
samples in the appropriate way prior to completing embedding.
An electrically cooled area to speed up setting of the tissue blocks. This may be a fixed
part of the embedding centre or purchased as a separate unit.
A timer control to switch the machine on and off at appropriate times in the day to suit
laboratory workflows.
The operator should be able to vary the temperatures of these heated and chilled parts
of the equipment to suit local needs. Frequently the equipment is supplemented by the
purchase of additional electrically heated forceps to permit the easy manipulation of
specimens and to reduce the carry over of specimen fragments from one specimen to the
next. The embedding area is the place in which the tissue is placed together for the
creation of tissue blocks. It is essential that the work area is kept spotless to ensure that
only the material from which the case is being worked upon is placed in the tissue block.
Embedding is the process of orientating and fixing tissue in a

hard media
TISSUE PROCESSING
Level
Competence
Can load and

unload tissue
processors with
tissue blocks
Can flush
machines to
Page 38 of 61
Assessed
competent
Assesor
Countersigned
by candidate
Level
Competence
Assessed
competent
Assesor
July 18,
2011
Countersigned
by candidate
prepare for a new

run
1
Can change
reagents on
processor
Can update audit

log documentation
for reagents
EMBEDDING
Level
Competence
Embed large 'slabs'
1
1
1
1
1
1
1
2
Assessed
competent
Assesor
Countersig
ned by
candidate
Read SOP
Select the correct mould
Top up wax
Put cassette on mold flat
Clean wax drains
Wipe work area clean
Insert bead
Orientate biopsies
Page 39 of 61
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Level
2
2
2
2
Competence
Assessed
competent
Assesor
Countersig
ned by
candidate
Orientate skins
Orientate tubes
Can change fuses on
embedder
Complete CP/H9C
ROUTINE SECTIONING
Sectioning is the part of the histology process in which microscope slide preparations are
made. The produced slide preparations are translucent and can vary in thickness from
0.1m to 50m. The thinnest sections are required for electron microscopy and the
thickest sections are required for neuropathology techniques.
MICROTOME TISSUE SECTIONS ARE PREPARED ON A

MACHINE CALLED A MICROTOME. THIS WORD COMES FROM
THE GREEK MICRO SMALL, TOME CUT.
Good section cutting is facilitated by tissue blocks being cut from a media which is about
the same hardness as the tissue. Wax embedding is not really suitable for tissue which is
very hard. Tissues which are very hard either need to be softened or embedded in a
harder media such as resin. The microtome is an essential piece of equipment in the
histology laboratory. Microtomes are available in a number of different forms. The rotary
microtome is by far the most popular, and can be semi automated. The sledge and
sliding microtome are popular in some laboratories for the simplicity of use and quality of
sections. Whatever the type of microtome, the basic function is the same, to prepare
tissue sections of a known thickness in a consistent manner.
For routine diagnostic specimens embedded in paraffin wax the objective is to cut a slice
from the tissue that is approximately one cell in thickness. In practice this means that
samples are routinely sliced (sectioned) at approximately 4m. Multiple sections are
often required from the same block and the microtome must be able to provide ribbons
of sections.
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Hard material such as bone may need a different approach to softer tissues. Brittle
material such as heavily keratinized skin or thyroid colloid may need softening prior to
sectioning; this can be done with water phenolic based reagents or surfactant containing
softening agents. Recent comparative studies suggest that softening agents containing
surfactants perform better on a cross section of hardened tissue types.
Each block contains many sections worth of material and it is important to go sufficiently
deeply into the block to obtain an appropriate cross section of material also it is equally
important not to waste material. It is quite possible that numerous additional tests will be
required. The way each block is sectioned depends on a number of factors; the type of
tissue, the size of the tissue or the clinical history. Some blocks will require a single
section for H&E, some require a number of sequential sections, to follow a lesion or tissue
element through the tissue, and this is known as serials. Some require a number of
sections distant to each other through the block to look at random parts sequentially, this
is known as levels. Looking at various sections through the material allows you to see the
clinical pictures in three dimensions. Often an H&E slide alone is required but there are
specimens which need multiple different types of stain to obtain a diagnosis e.g. liver
biopsy, renal biopsy.
WHY ARE MOST ROUTINE SECTIONS CUT AT 4M?
Tissue blocks need to be cut with care so that sections are deep
enough to reveal any pathology but shallow enough to allow
further sectioning.
Health and safety

Microtome blades are extremely sharp and it is easy to injure
yourself, particularly if concentration wanes. If you feel your
concentration flagging, take a short break and return later.
FACTORS AFFECTING SECTION CUTTING

THERE ARE A NUMBER OF FACTORS WHICH AFFECT THE QUALITY OF CUT
SECTIONS. THESE FACTORS INCLUDE
SHARPNESS OF THE CUTTING BLADE
RIGIDITY OF THE KNIFE AND SPECIMEN HOLDER
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HARDNESS OF THE TISSUE

BLOOD WITHIN THE TISSUE
THE COLDNESS OF THE BLOCKS
SHARPNESS OF THE CUTTING BLADE
RIGIDITY OF THE KNIFE IN THE SPECIMEN HOLDER
The cutting blade needs to be sharp and free from defects. Most laboratories use
disposable blades which are clamped within a knife holder. As more tissue is
progressively cut the knife becomes blunt. Any pieces of calcium can nick the knife
causing sections to score.
Hardness of the tissue
If the tissue is very hard it can cause the tissue to cut thick and thin. This is because the
difference between the hardness of the embedding media and the tissue causes a
speeding up and slowing down of block through the blade.
Blood within the tissue
Blood does not process well and dries out. When dry the blood cracks on cutting. Blocks
containing blood cut much better when they are soaked or cut from wet ice.
Coldness of blocks
Blocks need to be cool in most circumstances. The colder the block the harder the wax
will be, the harder the supporting matrix, the easier it is to cut thin sections. There is a
point that if blocks are too cold then the tissue itself becomes too brittle to section and
ribbon easily. This means that the integrity of the overall section is spoiled, the sections
may become chattered.
Special considerations when cutting blocks
Lymph nodes
These are very cellular specimens and so tend to look very crowded under the
microscope when sectioned at 4m. Far better cellular resolution is achieved if these are
sectioned routinely at 2m.
Renal biopsies
As with lymph nodes, the resolution of the glomerular basement membrane is much
improved if sectioned routinely at 2m.
Amyloid
Page 42 of 61
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2011
The demonstration of amyloid variants with Congo red solutions is improved if thicker
sections than usual are obtained. Ideally these should be sectioned at around a thickness
of 8m
Neuropathology sections
Again thicker sections are most suited for many of the tinctorial and metallic
impregnation methods used. Thicknesses of between 15m and 50m are frequently
encountered to be able to view and follow nerves through the section.
Microtomy
Material is processed to paraffin embedded tissue blocks, at this point key parts of tissue
have been sampled, fixed and processed so that all the water has been removed and
replaced with an embedding media. The embedding media is the same hardness as the
tissue. All these steps are to produce tissue blocks which are capable of having thin
sections cut, this is the process of microtomy. Microtomy is the process of producing thin
tissue sections from tissue blocks, using a specialist piece of equipment called a
microtome. Microtomes exist in a number of forms -rotary, sledge and sliding.
All equipment handled during the microtomy process should be kept scrupulously clean.
It is important that cells from one block are not transferred to the slides belonging to
another block. This form of cross contamination is known as carry over.
MICROTOMY EQUIPMENT
THE KNIFE
For routine paraffin sectioning traditional steel knives have almost completely been
replaced by disposable carbon steel blades. For all but the toughest of tissues they
provide a superior edge to steel knives and have also permitted the production of
consistent sections of high quality at thicknesses less than 4m which has improved the
morphological assessment of very cellular specimens. However the blades are not
inexpensive and so represent a significant drain upon the budgetary resources of a
department. In addition disposable blades do not require re-sharpening so the
acquisition of sharpening instruments is no longer necessary and the health and safety
considerations associated with the sharpening of steel knives has been avoided.
Knives lose their sharpness with use due to general wear and tear issue. Knives that are
blunt will score sections and eventually fail to ribbon blocks. If this happens the knife
should be sharpened or the blade disposed of and replaced which us expensive.
ROTARY MICROTOMES
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2011
In this design the tissue block and knife are held in the vertical plane. Turning the handle
(flywheel) one complete revolution advances the tissue towards the blade at whatever
thickness has been set. As the two make contact a sliver of wax, containing the tissue, is
shaved from the surface of the block. See figure 3.25
SLEDGE MICROTOMES
This version works similarly to the rotary microtome with the exception that both block
and knife are positioned horizontally and the block is slid backwards and forwards in this
plane making contact with the knife. As the block is slid back towards the operator on
runners (or a sledge) there an advance mechanism is operated which raises the block
towards the knife by the required section thickness.
SLIDING MICROTOMES
Unlike the previous two with this instrument it is the knife that moves towards the
stationary block in a reverse of the situation encountered with the sledge microtome.
Like the sledge both block and specimen are orientated in the horizontal plane. The blade
cannot be clamped at both ends as is possible with the rotary and sledge microtomes so
this instrument is not particularly suited for cutting harder material. Also the moving
blade makes it less satisfactory from a user's health and safety perspective! Whereas the
previous two would commonly be seen in routine histopathology departments the sliding
microtome is more suited to specialist applications, in particular for the sectioning of
nitrocellulose blocks in neuropathology facilities.
FREEZING MICROTOMES
The sample is frozen onto the cassette holder. The blade is then drawn over the tissue
sample to produce the section.
CAMBRIDGE ROCKER
Although not used in many establishments, the Cambridge rocker has had an important
role in the development on microtomy. The tissue block rocks on a stand against a blade.
The resultant sections are cut in an arc from the block. The simple design of the
Cambridge rocker has made them long lasting and easy to repair.
HEALTH AND SAFETY

MUCH MICROTOMY WORK INVOLVES THE USE OF KNIVES.
CARE MUST BE TAKEN TO ENSURE THAT THEY ARE
APPROPRIATELY GUARDED. WHEN KNIVES ARE NOT IN
USE THEY SHOULD BE COVERED OR IF FINISHED WITH
DISPOSED OF.
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THE WATERBATH
Sections are floated out onto waterbaths at a temperature just below the melting point of
the wax, often about 50oC. This allows folds in the sections to be relaxed out of the
tissue before being picked up onto microscope slides. Some tissues require adhesive
slides to maintain the tissue sections on the slides. The adhesives may be
electrostatically charged or be natural sticky products such as albumen. Figure 3.46
show tissue sections being floated out on a waterbath. The knife on a microtome needs
to be well clamped to obtain evenly thick sections.
The floating out is an important part of the process, sections need to be floated out for
the appropriate length of time, if they are floated out for too short a length of time then
sections may be creased if they are floated out for too long then sections may
disaggregate or disintegrate.
Health and safety

The waterbath is a potential source of cross contamination
between cases. To ensure cleanliness the waterbath should be
regularly skimmed of any debris using a tissue
SLIDES
It is important when you prepare good quality sections that you dont spoil all your effort
by the use of inappropriate microscope slides. It is important slides should be clean and
free from specs of dust so that the tissue section is not disrupted or torn. Commonly
slides are either uncoated or coated to aid adhesion of sections on to the slide. The
coating of slides can be from a number of sources, such as, albumin (egg white), 3aminopropyltriethoxysilane (APES) and positive charged coating.
MICROTOMY
Level
1`
1
Competence
Assessed
competent
Assesor
Countersigned
by candidate
Has read SOP

Can trim large
blocks
Page 45 of 61
20th
September
2011
1
1
1
Can cut large

block section
with no
artefact
Can use
microtome
safely
Can float
sections on
waterbath
Can write
slides with
correct patient
details and
own symbol
Can trim, cut
and float 20
large blocks in
1 hour
Can cut and
orientate
ribbons
Can cut mega

blocks
Can cut and

orientate levels
Has completed
CP/H10c
Can trouble
shoot
microtomy
problems
Can dismantle
and set up
microtome
CRYOTOMY
Cryotechniques are laboratory methods using tissues that have been frozen solid.
Freezing techniques may be used for a variety of reasons, speed, and preservation of cell
enzymes or avoidance of chemical fixatives due to the interference with the method
under investigation. Tissues may be frozen by liquid nitrogen, card-ice ( solid CO 2),
electrical plates or ozone depleting fluorocarbon spray. It is important to freeze tissue
quickly and evenly to prevent ice crystal artefact, see box for more details.
Page 46 of 61
July 18,
2011
ICE CRYSTAL ARTEFACT

Ice crystal artefact is when tissues are damaged as they are frozen by ice crystals in their
tissue. The water within the tissue freezes as large crystals which tear at the tissue when
they freeze. Figure 3.x shows an H&E frozen section with ice crystal artefact. Note how
the tissue has large ragged holes in it.
Frozen sections are cut on a cryostat (see figure 3.25). Cryostats are microtomes
which are housed in freezer units. They are chilled to about -20 oC. The front housing has
a glass panel which closes off the unit from the warm air of the room. Tissue handled in
cryostats may contain high risk micro-organisms or viruses such as TB, HIV or Hepatitis
B. It is therefore essential that a cryostat can be decontaminated and fumigated to
disinfect the microtome and cooling chamber. Frozen sections are also more difficult to
cut thinly i.e. <5m, and do not allow for the examination of material in as much detail
as paraffin processed tissue. In other words the morphological features are not as well
preserved in frozen sections compared to paraffin processed tissue.
FROZEN SECTIONS
Frozen sections are a way of reporting histology samples rapidly for example, when a
patient is in theatre and the results may inform the action the surgeon takes next.
Sectioning of these samples is made possible by freezing the tissue to provide a firm
supportive matrix from the water within the tissue frozen as ice. The tissue may be
cooled by a Peltier plate ( a bimetallic strip which has a current passed through it),
freezing spray, card-ice (solid carbon dioxide) or liquid nitrogen. Sections are usually a
few microns thicker than usual at about 5m. Frozen sections are received when the
patient is operated on and the course of the surgical intervention is dictated by the
histology. The surgeon often wants to know whether a mass is malignant or not, if it
needs removing or does the patient require surgical palliation? Patient results from frozen
section specimens should be phoned back to the operating theatre within 20 minutes of
receipt. Common frozen sections are taken from abdominal cavity nodules or lymph
nodes looking for the presence of cancer.
Case Study: -Frozen section
A surgeon is about to remove a skin lesion from close to the
patients eye. The aim of the operation is to remove the lesion
but not too much surrounding skin so that the cosmetic change
to the patients face is minimal.
Specimens are sent for frozen section to assess removal of the

lesion.
Page 47 of 61
20th
September
2011
Frozen sections provide a quick way to cut, stain and report a

section in twenty minutes. If this is so then why are not all
sections cut this way? Frozen sections provide a very good way
of providing a single section but it would be too time- consuming
to cut all sections this way as each individual slide takes longer
to make. The amount of cellular detail is less in a frozen section,
and the size is limited to about 1cm in diameter for most tissues.
It is also worth noting that fatty tissue cuts quite poorly on a
cryostat due to the fact that it needs to be much colder than
other tissues types.
CLARKES FIXATIVE AN ALCOHOL AND GLACIAL ACETIC ACID SOLUTION WITH RAPID
FIXATION BUT POOR TISSUE PENETRATION. THIS FIXATIVE IS IDEAL FOR FIXING FRESH
SECTIONS ADHERED TO A SLIDE
Formalin fixation and paraffin wax processing takes several hours to complete even for
very small diagnostic samples. The use of frozen sections achieves a thin section suitable
for staining and microscopic examination within minutes.
Some enzymes are labile to the chemicals used in conventional paraffin wax processing.
Frozen section processing removes the need for these chemicals so that these enzyme
sites can be demonstrated. Many enzyme histochemistry methods demonstrate
enzymes which are damaged by formalin fixation, therefore frozen sections must be used
with these samples.
URGENT FROZEN SECTIONS

Frozen sections may be requested by surgeons prior to, or during an operation. The
purpose of an urgent frozen section during an operation is to determine the pathology of
suspicious tissue found during surgery. In the main the question the surgeons want to
know the answer to is: Is the tissue malignant?
INVESTIGATION OF INFLAMMATORY SKIN DISORDERS

Patients may develop rashes and bullae which require investigation. Small skin samples
from the edge (not the blisters themselves) are taken and sent to the lab fresh, or in a
Page 48 of 61
July 18,
2011
non fixative solution. Michel's transport media is often used. The differential diagnosis
includes pemphigoid which has linear immunoglobulin deposited at the dermo-epidermal
junction. Investigation of these immunoglobulins is best done with immunofluorescent
techniques. Immunofluorescent techniques use antibodies labelled with fluorescent dyes
to mark antigens deposited in the tissue, it is used in preference to other methods due to
good contrast which these tests give. The dark background of the immunofluorescent
test gives excellent contrast with the brightly staining antigens.
The number of antibodies used in immunofluorescent technique is limited often to IgG,
IgA, IgM, C3c and fibrinogen.
Certain lab tests are prohibited by the use of formalin, this is so with
immunofluorescence as formalin fixation causes auto fluorescence under UV light.
CRYOTOMY
Level
Competence
Can cut frozen

sections at 6m
thick in a timely
manner
Can stain frozen

sections by hand
Can change a
blade on the
cryostat
Can
decontaminate
cryostat
Assessed
competent
Assesor
Countersigned
by candidate
STAINING AND COVERSLIPPING

STAINING
Page 49 of 61
20th
September
2011
When sections are mounted and heat fixed on to slides they are available for staining.
Staining follows one of a number of strands
General morphology stains such as H&E
Special stains looking for specific tissue elements identified by chemical interaction
between dyes and the tissue e.g. elastin stains ( see chapter 4 for more details)
Immunocytochemistry. The interaction of antibodies and tissue elements.
Molecular techniques, investigations into the DNA and RNA of the cell.
The haematoxylin and eosin stain
The prepared sections mounted on the microscope slides are translucent and require
staining. The H&E stain is the first stain performed on most material when it enters the
laboratory. It has the advantage over other stains that it provides a good staining of the
majority of tissue components and is cheap. Most importantly, it gives lots of information
about the cell nucleus. The eosin stain gives a good demonstration of connective tissue
and cytoplasmic elements. H&E stained slides provide sufficient diagnostic information to
be able to report 80 - 90% of all cases, in the remaining 10 - 20% cases further tinctorial,
immunocytochemistry or molecular tests are required.
Haematoxylin
Haematoxylin is a dye originally derived from the logwood tree, a tropical hardwood tree
found in Central America. Haematoxylin itself stains tissue poorly and for effective
staining to take place it needs to be oxidised and linked to a mordant. For routine
staining an alum mordant is used. You have read about this in chapter 2
Staining may be either progressive or regressive. Progressive staining is where the

tissue is stained until the point that all tissue elements are stained correctly. This requires
fine control of the reagents. Regressive staining is where the sections are over stained
and excess stain is removed by using a dilute acid. This is known as regressive staining.
Haematoxylin exists in many different forms. Common haematoxylin solutions used in

the laboratory are
Carazzis Haematoxylin
Ehrlichs Haematoxylin
Gills Haematoxylin
Harris Haematoxylin
Page 50 of 61
July 18,
2011
Mayers Haematoxylin
EOSIN
Originally eosin was derived from crushed beetles, now manufactured synthetically. The
eosin molecule is negatively charged and binds to the positively charged components
within the cytoplasm. Eosin solutions are either prepared in alcoholic or aqueous
solutions. The synthetic form is often combined with acetic acid and/or calcium chloride
to enhance the staining.
STAINING MACHINES
The staining of routine sections in most laboratories is no longer carried out by hand and
automated staining machines are used . The purpose of an automated staining machine
is to hold the slides in a series of reagents in order for a set period of time. Automated
staining machines save a lot of laboratory time but do require some regular routine
maintenance. Many of the reagents used by the staining machines are harmful and
vapours from these machines need to be controlled. Fans and activated charcoal filters
are used to do this.
Most staining machines available on the market allow the storing and use of multiple
programmes. This allows for slides of different material to be stained for different lengths
of time.
Types of automated histology stainers
Linistainers consist of a basic chain (very similar to a bicycle chain) which is attached to
an electric motor which runs at a constant speed. The length of time in each reagent is
determined by the width of the staining pot used i.e. wider staining pots have the slides
immersed in them for a longer period of time.
There is an end trough containing xylene where the stained sections are stored prior to
coverslipping.
XY stainers are histology stainers with a robotic arm which places the slide racks in
each staining pot for a fixed length of time. The staining time is programmed and is not
dependent on the width of the staining pots as with linistainers. Multiple racks of slides
can be stained at any one time. XY stainers provide a flexible approach to staining
together with a rapid throughput of slides. Figure 3.27 shows an XY stainer linked directly
to a coverslipper so that the unstained slides can be placed on the machine and a
coverslipped end product is then available.
Page 51 of 61
20th
September
2011
STAINERS FOR SPECIAL STAINS

Although most laboratories still perform special stains manually there are now a number
of automated processors available for handling slides. Machines for special stains contain
a number of reagent bottles which are slotted into the machine and slides are immersed
or covered in the solution for a set period of time.
COVERSLIPPING
Once the slides are stained, the preparations need to be made permanent by putting a
protective covering over the tissue section, usually either glass or plastic film. Coverslips
are thin pieces of glass used to cover tissue sections and protect them from damage. The
protective layer is between 1.0 and 2.5mm in thickness adhered by mountant a special
type of glue. The mountant used needs to be the same refractive index as the glass slide
and he coverslip. This helps prevent refraction artefacts.
Coverslipping can be done either by hand or automatically. There are a number of basic
aims for coverslipping
To place a coverslip over the section
To ensure that there is sufficient mountant so that there is no leeching back of mountant
That there is not too much mountant that the top of the coverslip becomes encrusted
with mountant.
That there are no air bubbles trapped under the coverslip
ALL HISTOLOGISTS SHOULD BE ABLE TO HAND COVERSLIP. THE PROCESS INVOLVES

PLACING MOUNTANT ON A COVERSLIP AND THE SLIDE AND SECTION ADDED TO THE
TOP OF THIS.
AUTOMATED COVERSLIPPERS
Automated coverslippers have been developed to speed up the coverslipping process in
the lab. They use either glass coverslips or plastic strips on tissue sections.
AUTOMATED GLASS COVERSLIPPERS

Glass coverslippers place individual glass coverslips over the tissue section. The
mountant is usually added to the slide and the coverslip flexed by the machine to
squeeze any air bubbles out. Coverslips need to be sufficiently robust (Usually 1.5mm
thick) to withstand a small amount of flexing.
TAPE COVERSLIPPERS
Page 52 of 61
July 18,
2011
The principle of the tape coverslipper is that the tape is unrolled over the section then
cut. This is a quick process but some laboratories have had problems when archive
sections have had their tape coverslips fall off with time. .
STAINING AND COVERSLIPPING
Level
Competence
Can hand stain a

H&E
Can hand
coverslip
Can change the

reagents on a
staining machine
Can load a rack

on to the stainer
and coverslipper
Assessed
competent
Assesor
Countersigned
by candidate
INTERNAL QUALITY CONTROL FOR ROUTINE H&ES

Internal quality control is the system followed in the laboratory to maintain the quality of
the work. This will involve ensuring that the stains carried out are checked for their
specificity and accuracy of staining. As a minimum the routine haematoxylin and eosin
stain should be checked at the start of each day. In most labs this process is automated
so this involves the checking of the first section stained using the staining machine. The
IQC slides produced are stored for review and audit.
BLOCK CHECKING
After sections are cut and stained the blocks are checked against the slides cut so that
the shape of the sections is checked and the patient name and case number are
checked.
MICROSCOPE CHECKING
Page 53 of 61
20th
September
2011
Each slide, or a selection of slides from a case, are checked under the microscope for
staining quality and for quality of microtomy. Sections which are of poor quality are
rejected and a replacement section is cut.
The key items investigated when microscope checking slides are
Is the fixation OK?
Is the section suitable
Deep enough?
Representative?
Are there any cutting defects? Creases? Folds? Scores?
Is the staining of good quality?
Is the coverslipping OK? Are there any air bubbles?
Is the presentation OK? Is the section central?
Are the correct patient details on the slide?
Checking the request form

The request form is checked to see that the tissue seen down the microscope matches
that requested on the card. The number of blocks taken at cut up and the number of
pieces are documented on the request card and these details are checked as well. Once
all the checks have been satisfactorily carried out then the slides can be passed to the
pathologist for reporting.
Any discrepancies are noted and logged using the department's error logging system.
INTERNAL QC
Page 54 of 61
Level
Competence
Able to Qpulse
quality issues
Can generate
a list and print
labels
Can block
check
Can check
details on form
against slides
and blocks
Can recognise
microscopicall
y skin,
endomyo,
breast, liver,
kidney, cervix
and colon
Can check
quality if stain
Can identify
artefact
Has read SOP
Can approve
and close off
worklists
Assessed
competent
Assesor
July 18,
2011
Countersigned
by candidate
Page 55 of 61
20th
September
2011
Level
Competence
Improved
tissue
recognition
biopsies (oral,
oesophageal,
cardiac
antrum,
jejunum,
duoddenum,
colon)
Can trouble
shoot staining
quality failures
Can check
orientation of
tissue
(e.g.skin)
Assessed
competent
Assesor
Countersigned
by candidate
Assessed
competent
Assesor
Countersigned
by candidate
MICROSCOPY
Level
Competence
Can set the

microscope up
for Kohler
illumination
Can change
microscope
bulb
Can clean
lenses
Page 56 of 61
July 18,
2011
Completion of
CP/H62C
SPECIAL STAINS
When tissue sections are first prepared, they need to be stained in order to see any detail
of their structure. This is because fixed, processed tissue lacks contrast, and looks the
same colour despite having numerous entities within them. In order to see the different
structures in tissues, a variety of coloured dyes and stains can be used to emphasise the
different structures present. These stains have evolved over many years, and in the
modern diagnostic histology laboratory there are dozens of methods available to colour
many entities in contrasting colours.
Most of these methods are based on chemical principles, whereby structures of a specific
chemical nature can be selectively stained with the appropriate dyes. Often, two or more
dyes can be used to show vivid contrast of the variable structures within the tissue.
Many of the dyes used today in histological demonstration were first produced over a
hundred years ago, not for histology tissues, but in the colouring of fabrics such as
cotton, when the textile industry was developing. Many natural dyes from plants were
used until the late 1800s when synthetic dyes were introduced. Dyes such as madder
date back thousands of years. Other dyes including cochineal, indigo, logwood
(haematoxylin) are still used today.
The most common staining method in use today is Haematoxylin and Eosin (H&E), and is
used as a routine stain in most histology laboratories worldwide to show the general
morphology of tissues. The results show nuclei as blue, black or purple, with other
structures such as connective tissue, erythrocytes and cytoplasm stained in a contrasting
red or pink.
PASD
Competence
Date
assessed
Assess
or
Assessors
signature
Candidates
signature
Can select appropriate

control material
Can prepare slides stained
with PASd
Can label control slides
appropriately
Page 57 of 61
20th
September
2011
Competence
Date
assessed
Assess
or
Assessors
signature
Candidates
signature
Date
assessed
Assess
or
Assessors
signature
Candidates
signature
Date
assessed
Assess
or
Assessors
signature
Candidates
signature
Can check stained slides for

quality
PAS
Competence

control material
with PAS
appropriately
quality
GROCOTT
Competence

control material
with Grocott
appropriately
quality
HVG
Page 58 of 61
Competence
July 18,
2011
Date
assessed
Assess
or
Assessors
signature
Candidates
signature
Date
assessed
Assess
or
Assessors
signature
Candidates
signature
Date
assessed
Assess
or
Assessors
signature
Candidates
signature

control material
with HVG
appropriately
quality
PERLS
Competence

control material
with Perls
appropriately
quality
RETIC
Competence

control material
Page 59 of 61
20th
September
2011
Competence
Date
assessed
Assess
or
Assessors
signature
Candidates
signature
Date
assessed
Assess
or
Assessors
signature
Candidates
signature

with retic
appropriately
quality
MSB
Competence

control material
with MSB
appropriately
quality
MASSON TRICHROME
Competence
Can select appropriate control

material
Page 60 of 61
Date
assessed
Assess
or
Assessors
signature
Candidates
signature
Competence
July 18,
2011
Date
assessed
Assess
or
Assessors
signature
Candidates
signature
Date
assessed
Assess
or
Assessors
signature
Candidates
signature
Can prepare slides stained with

Masson Trichrome
appropriately
quality
ZN
Competence
Can select appropriate control

material
Can prepare slides stained with
Masson Trichrome
appropriately
quality
Page 61 of 61

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HISTOLOGY COMPETENCY MANUAL

Often used interchangeably with sample, describes the material

The process of tissue preservation with chemicals. Fixation acts to

The process of cellular degradation due to micro-organism

The removal of water from tissue and replacing it with another

The document supplied with tissue samples giving details of the

The chemicals and solutions used in the histology laboratory

The standard staining method used in histology. It is able to show

HISTOLOGY COMPETENCY MANUAL

A range of stains used to identify specific tissue elements. Usually a

A range of antigen antibody related tests where antibodies are

Human tissues do not look coloured under the microscope by

A thin piece of tissue. This is usually one or two cells thick,

The container in which a specimen is received. It should be labelled

A large tissue sample removed for analysis. It may be a kidney, a

HISTOLOGY COMPETENCY MANUAL R1

A skin biopsy with the lesion completely removed There should be

A machine which cuts sections. The basic operation is a knife and a

A microtome in a refrigerated unit. The cutting temperature is

This word is used interchangeably with pieces of tissue used in

Larger plastic cassettes containing tissue. They are big enough to

A machine which processes tissue. Tissues are moved through a

The material which tissue is held within for cutting sections.

An area of suspected pathology, that may be well or ill defined,

HISTOLOGY COMPETENCY MANUAL

The minimum specimen information should be

What the sample is

WHEN SPECIMENS ARE RECEIVED INTO THE LABORATORY IT IS IMPORTANT TO CHECK

What patient information is required to accept a specimen in a histology

HISTOLOGY COMPETENCY MANUAL R1

SUBSEQUENT TESTS WITHIN THE HISTOLOGY LABORATORY FOLLOW DEPENDING ON

Full clinical information helps the laboratory to make a full diagnosis

HISTOLOGY COMPETENCY MANUAL

ENDOSCOPIC BIOPSIES ARE BIOPSIES OBTAINED BY USING A FLEXIBLE TUBE

Histology takes time

HISTOLOGY COMPETENCY MANUAL R1

Urgent samples fall into two groups

URGENT FIXED SAMPLES

What clinical circumstances might indicate an urgent specimen?

explain the purpose of the procedure

accurately maintain records

HISTOLOGY COMPETENCY MANUAL

demonstrate completion of procedure

1. State the purpose of the specimen reception procedure

2. State the health and safety risks of formalin

3. State the use of Michels transport media

4. Describe the specimen acceptance / rejection policy

5. Describe how specimens are allocated to consultants

6. Describe the purpose and limitations of frozen section diagnosis of histology

7. Why are specimens sometimes sent to referral centres

8. What constitutes a high risk sample? How should they be handled?

HISTOLOGY COMPETENCY MANUAL R1

10. Why are specimens of the same type kept separate?

11. Why are gastro-intestinal specimens labelled in anatomical order?

12. Why are records of rejected specimens kept?

HISTOLOGY COMPETENCY MANUAL

IBMS PORTFOLIO CROSS-REFERENCE

SPECIMEN DISSECTION ROOM

HISTOLOGY COMPETENCY MANUAL R1

Autolysis is the process of cellular degradation by the release of intracellular enzymes

HISTOLOGY COMPETENCY MANUAL

Kill cells quickly and evenly