the steps from dissection of the tissue to producing a section on a slide as
provided for this practical and as would be carried out in a hospital lab.
A section on a slide must be fixed, the original sample is trimmed,
transferred into a label plastic and stainless-steel carrier. The carrier is
placed in the automatic processor. In tissue processing,
the water in the tissue is replaced with modern paraffin wax to remove the
water, the tissue samples go through increasing strength of alcohol solutions.
The tissue is dehydrated gradually to avoid excessive shrinkage. The tissue is embedded, removed and orientated as
required in a metal mould which has been filled with molten wax. The mould is
topped up with extra wax and placed onto the adjacent cold plate, the wax
solidifies then removed. The tissue is block is clamped into the microtome for sectioning. The knife, inserted and
clamped in the holder, the stage is eased towards the block. With the required
thickness, routinely 5 microns, the handle rotation advances the block by 5
microns and lowered down past the knife to produce a section. Due to compression,
the tissue sections are slightly wrinkled, but most folds can be removed by
floating up the ribbon or individual sections on a warm water bath. Sections
are collected up onto a microscope slide and dried in a 40o heat.
are the majority of samples sent to the histopathology laboratory as ‘fixed’
Specimens must be fixed because Fixation
is an essential preparation step prior to microscopic examination with the
objective in prevention of decay and preservation of cells and tissues. It stops
the activity of enzymes and kill microorganisms. Furthermore, hardens the
specimens but keeps sufficient structure for light microscopic examination.
Fixation alters tissue by stabilisation of protein to resist further change. It
works by converting tissue from soluble to insoluble: preventing loss during
the processing stage. Functionally, fixatives are to kill tissue preventing
autolysis; to maintain structure and to bring out refractive indexes to
increase visibility and contrast. There are several types of fixative including
additives where the fixatives combine with the tissue protein making them
insoluble. Non-additives, organic compounds that act on the tissue without
chemically combining with it; coagulates that create a mesh ball like tissue network
that allows solutions to more easily penetrate the section; and non-coagulants,
these create a gelatinous environment making it more difficult for subsequent
solutions to penetrate the tissue.
direct or indirect labelling used in this practical and how did you decide this
from the protocol?
The use of a second antibody is understood
to be indirect labelling. This is where two different antibody reagents are
needed to be able to visualise the target. For example, a specific antigen
primary antibody, and an antigen secondary antibody. Routinely, the second
antibody is associated with the detection of components like an enzyme, a
Within the protocol, 3-amino-9ethyl-carbazole (AEC) was used as a second antibody as a
detection reagent. Indicative of a secondary labelling. Indirect labelling
requires two different antibodies reagents to be able to visualise the target.
However, it is not as simple, as it needs an addition of blocking steps and control which we did not have in the protocol.
The addition of Streptavidin and
peroxidase solution on top of the biotinylated secondary antibodies
amplifies further the ability for those substances to bind to more secondary
antibodies. Also, using chromogen for the development of colour reaction
because of the high signal in the method. The detection using chromogen is
efficiently sensitive compared to fluorescent.
are the main differences between the detection of a protein by a monoclonal antibody
and a polyclonal antiserum?
Polyclonal antiserum has many epitopes present
on antigens. Those antibodies are not specific; therefore, they have the
recognition capacity of multiple epitopes on many antigens. This capacity
increases the signal released by target proteins due to the antibody binding to
more than one epitope. Compared to monoclonal antibodies, polyclonal detection
is not as sensitive to antigen as in monoclonal detection. However, the
detection in polyclonal is more potent because of multiple epitopes on antigen.
In contrast, monoclonal antibodies detection of target protein is highly
specific because monoclonal antibodies are identical and highly specific to the
epitope found on the antigen. As a result, there is a reduction of background
noises and cross-reactivity from other proteins, giving an efficient
might antigen retrieval be needed in immunocytochemistry and what are the main
Antigen retrieval is needed because of the
cross-linking fixative from paraformaldehyde or formalin, the crossways can
sometimes mask the epitope. Epitope, a specific sequence of amino acids that
bind onto the antigen binding site of that immunoglobulin of the antibody. Once
the binding process takes place, the antibody antigen complex is formed. To reserve
the cross-links, the antigen retrieval technique is used; also known as epitope
The two most common methods of antigen
retrieval are the heat induced retrieval as well as the enzymatic retrieval.
The heat induced retrieval dissection on the slides are heated and buffered
with two main buffers which are sodium citrate at a low pH of 6 or EDTA buffer
at a higher pH of 9. The sections in the solutions are then heated and this is
accomplished in a pressure cooker, additionally microwave. For enzymatic retrieval,
tissues are incubated with enzymes trypsin, pepsin etc. to reserve cross-links
and make the epitope more accessible.
the intensity of staining scored in a histopathology lab? How can this be
standardised to allow comparison between different patients and hospitals?
The intensity of staining is scored using
the Allred scoring system on a scale of 0 to 3. 0 for negative; 1 for weak; 2
for intermediate and 3 for strong intensity. The scores are obtained manually
or via an automated staining software. The intensity depends on many variables
such as incubation time, temperature and the acidity level of chemicals. A high
percentage of global pathology hospitals still utilise manual and for some,
automated staining system. To be able to standardise the scoring, during
staining, controls for the temperature, pH, incubation length and the number of
antibodies will have to be the same across all hospitals and for all the
patients. One way to improve this is to use the automated staining system
instead of the manual method. The system provides consistent results with less
repeats compared to the manual method. This therefore gives a fair comparison
of results across hospitals and patients as well as improving confidence in
diagnosis in pathologists.
this practical a control slide was not used. In a hospital lab what controls
might you consider using and what is the importance of control slides?
Control slides that might be considered
are negative, positive or internal negative and positive tissue slides.
Internal positive indicate a successful immunohistochemistry results. However,
it is more reliable for evaluating the appropriate sensitivity. Internal
negative can be very informative of the specificity of primary antibody. The
external positive and negative are the main tool to monitor the quality and
consistency. They can indicate the sensitivity of immunohistochemistry assay.
Control slides are important it can affect how staining is interpreted;
indicate whether the staining protocol was carried correctly. They give a
direct comparison on whether technicians variations have occurred and that the
reagents are fully functioning. More often used to demonstrate that the
immunohistochemistry assay was outstanding and able to show the target of