Discuss level of disease diagnosis in histopathology laboratory

There are three levels of disease diagnosis in histopathology laboratory. These levels
are based on the techniques used that ranges from basic to advanced techniques. Has
the levels advances the easier and the faster to produce the result as requested by the
pathologist.

The following paragraphs will discuss the levels of disease diagnosis in histopathology
laboratory based on tissue handling and the technique used for each level.

ROUTINE DIAGNOSTIC PROCEDURES

This diagnostic procedures is based on tissue morphology to provide diagnosis. After

the tissue sample removed from the patient, a series of physical and chemical
processes must take place to ensure that the final microscopic slides produced are of a
diagnostic quality.
Fixation is the first step of any histological laboratory technique. It is the process by
which the cells in the tissue are fixed in a chemical and physical state, and all the
biochemical and proteolytic activities within the cells are prevented so that the cells or
tissues can resist any morphological change or distortion or decomposition after
subsequent treatment with various reagents. The fixation helps to maintain the tissue
nearest to its original state in the living system. Fixation done in order to
 To preserve the tissue nearest to its living state
 To prevent any change in shape and size of the tissue at the time of processing
 To prevent any autolysis
 To make the tissue firm to hard
 To prevent any bacterial growth in the tissue
 To make it possible to have clear stain
 To have better optical quality of the cells
Formaldehyde, as 10% neutral buffered formalin (NBF) is the most common fixative
used in diagnostic pathology. The commonest way of fixation in the routine procedures
is Immersion fixation. In this technique the whole specimen is immersed in the liquid
fixative. Formaldehyde in aqueous solution combines with water to form methylene
hydrate, a methylene glycol:

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CH2O H2O =OHCH 2OH
In a long-standing position, this methylene glycol may further react with water molecules
and form a polymer known as polyoxymethylene glycol. This again depolymerized in
methylene glycol in a neutral buffer system. Formaldehyde reacts with various side
chain of the protein and forms hydroxymethyl side group. These compounds are highly
reactive and subsequently cross-linking occurs by forming a methylene bridge. This
preliminary reaction of hydroxymethyl side chain is the primary reaction, and the
subsequent intermolecular and intramolecular cross-linking of the molecules occurs as
a slow-growing process. This ultimately produces an insoluble product. The formalin
can be removed from tissue by prolonged washing. However, once Methylene Bridge is
formed in the tissue, the reaction is stable, and it is difficult to remove formalin from the
tissue. Formaldehyde also reacts with the nucleic acid by reacting with the amino group
of nucleotides.
The next step after fixation is the processing of tissue. This is also a very important step
because poor processing of tissue may significantly affect the section cutting and
staining. Proper handling of tissue specimens is critical to ensure that an accurate
diagnosis is obtained from patient tissue samples. Whilst technological advances have
streamlined processing, the principle steps remain the same: fixation, dehydration,
clearing and infiltration.
Aims of tissue processing: The basic aim of tissue processing is to provide sufficient
rigidity to the tissue so that it can be cut into thin section for microscopic examination.
Principle of processing: In tissue processing the water within the tissue is removed, and
another medium (usually paraffin wax) is impregnated in the tissue that provides the
adequate support to the tissue. Therefore the essential steps in tissue processing
1. Dehydration: In this step water is removed from the tissue. Water is immiscible with
wax, and therefore to infiltrate the tissue with wax, it is necessary to remove water.
2. Clearing: This is needed to clear the dehydrating agent and to facilitate the transition
of dehydration and impregnation stage. The clearing substance is usually miscible to
both dehydrating agent and impregnating medium.
3. Infiltration and impregnation: In this stage, the tissue is infiltrated with a supporting
medium which is suitable to provide adequate rigidity of the tissue to make thin section.

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  Fixation – prevents autolysis and stabilizes tissue to maintain cellular structure.
 Dehydration – removes water and unbound fixative from the tissue.
 Clearing – displaces dehydrating solutions, making the tissue components
receptive to the infiltrating medium.
 Infiltration – permeates tissue with a support medium.
 Embedding – orientation of the tissue sample in a support medium to create a
tissue “block” suitable for sectioning.

After embedding the tissue and preparing the block, the next step is microtomy. So the
word “microtomy” means to cut the tissue in thin sections. For successful microscopic
examination, it is necessary to have thin sections of the tissue by microtomy. A
microctome is the main instrument by which we cut the embedded tissue in the paraffin
block as thin section. The different types of microtomes in the traditional histology
laboratory but the commonly used in routine diagnostic procedures is a rotary
microctome. The cutting blade is kept in horizontal position, and the block containing
tissue moves up and down with the help of rotatory handle attached with the microtome.
In each 360° rotation of the wheel handle, the block moves down followed by up, and
the tissue is cut as thin ribbon. This microtome has the option to be semi-automated or
automated with the adjustment and control of the movement of the block and angle of
the knife.
The next step in routine diagnostic procedures is staining and the most commonly used
staining technique in routine diagnostic procedures that is used for general tissue
morphology is Haematoxylin and eosin which is a combination of two dyes. The
principle is based on two stains haematoxylin which stains the cell nucleus blue-black,
showing a good intranuclear details and eosin stains the cell cytoplasm and most of
connective details fibers in varying shades and intensities of pink, orange and red. The
rationale behind H & E is that it shows a very detailed view of the tissue by staining cell
structures including the cytoplasm, nucleus and organelles and extra-cellular
components hence achieving diagnostic purpose for routine diagnostic procedures.

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Commonly used H & E stains used in routine diagnostic procedure are Ehrlich’s,
Mayer’s, Harris’s, Gill’s, Coles and Delafied’s. The following are the steps in H&E
staining:
 The first step is to take the tissue to water thus deparaffinse section in xylene for
10-20 minutes.
 Rehydrate section in 100% alcohol 1-2 minutes and 95%alcohol for 1-2 minutes
 Rinse in tap water
 Rinse in distilled water
 Stain with haematoxylin for 3 minutes
 Wash in tap water
 Differentiate section with 1% HCL in 70% alcohol 1-2 dips and check under
microscope.
 Wash slides in running water
 Stain slides in eosin for 1-4 minutes
 Dehydration and differentiation (95% alcohol 5-6 dips and 100% alcohol 5-6 dips)
 Clear slides in xylene 2 times
 Most slides with mounting media.

The slides are now ready to be read by the pathologist

SPECIAL DIAGNOSTIC PROCEDURES

Special diagnostic procedures uses methods or techniques that answers questions or
provide information which routine diagnostic procedure failed to provide. It uses
techniques which demonstrate special features such as fungi or particular cell product.
The rationale of carrying out special diagnostic procedures is that it covers a wide
variety of methods or techniques that may be used to visualize particular or special
tissue structures, elements, or even microorganisms which cannot be visualized by
routine diagnostic procedure. The following are tissue elements and molecules that can
be demonstrated by special diagnostic procedures:
 Proteins and amino acids
 Carbohydrates

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  Lipids
 Nucleic acids
 Minerals
Techniques used in this diagnostic procedures are called special stains. All the
specialized stains are based on the affinity of the tissue elements, structures to
chemical reaction and bond formation with the use of various dyes and reagents. The
tissue sample can either be a new sample meaning a fresh sample or a tissue block
from routine diagnostic procedure. In this assignment I will consider the tissue block that
has been processed from routine diagnostic procedure.
There different special stains used in this diagnostic procedures, for this assignment I
will only discuss two namely Periodic Acid Schiff (PAS) and

Periodic acid Schiff

This is the most versatile and widely used technique for the demonstration of
carbohydrates or glycoconjugates.
The principle of PAS is based on upon the reactivity of free aldehyde groups within
carbohydrates with the Schiff reagent to form a bright red/magenta end product. Firstly
periodic acid will oxidize hydroxyl group (OH) of the carbohydrate molecule to aldehyde
(CHO) group. Then these aldehyde groups react with Schiff’s reagent to form a
magenta-coloured compound.

Feulgen Stain
This stain is specific for DNA and it demonstrates sugar deoxyribose. This is particularly
helpful for DNA ploidy examination.
Basic Principle In the presence of acidic environment (hydrochloric acid treatment), the
purine ases of the DNA molecule are detached from the deoxyribose. DNA molecule
becomes apurinic; however sugar-phosphate backbone of DNA is preserved. The
aldehyde group of the sugar molecule is exposed, and this group subsequently binds
with Schiff’s reagent to impart colour. This hydrolysis part is the most vital part of this
stain. DNA + Hydrochloric acid → Exposed aldehyde group of deoxyribose
Aldehyde group + Schiff’s reagent → Reddish purple colour

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Application Feulgen stain is particularly helpful for DNA ploidy examination.

ADVANCE DIAGNOSTIC PROCEDURES

Advance diagnostic procedures uses methods or techniques that answers questions or
provide information which special diagnostic procedure failed to provide. It uses
techniques which identifies cellular or tissue constituents, antigens, by means of
antigen-antibody interactions. Advance procedure are requested by the pathology either
as a primary diagnostic procedure or when the pathology has failed to make a clear
diagnostic decision with special diagnostic procedures.
Immunohistochemistry (IHC)
A technique for identifying cellular or tissue constituents, antigens, by means of antigen-
antibody interactions. The site of antibody binding is identified either by direct labeling of
the antibody or by use of a secondary labeling method.
The principle of immunocytochemistry is to demonstrate the specific antigen in the cell
by applying the corresponding antibody to have antigen-antibody reaction. The antigen
contains an epitope or antigenic determinant site that evokes specific immunologic
response to develop antibody. The antigen epitope site and antibody-binding site have
complementary geometrical and chemical features. This is responsible for the antigen-
antibody reaction. This antigen-antibody reaction is further visualized by attaching
certain label to the primary or secondary antibody.
It is not possible to detect antigen-antibody reaction by light microscope. Therefore
suitable detection or visualization system is necessary for the demonstration of such
reaction. There are different types of detection systems or techniques in this assignment
I will focus on four, system or technique as discussed below:

Direct Method In the direct method, the primary antibody is directly tagged with an
enzyme or fluorescence. The antibody should be specific for the particular antigen
otherwise non-specific staining may occur.
Advantage:
 Rapid and simple method.

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Disadvantages:
 Different primary antibody should be labelled differently for the antigen.
 Low sensitivity.
Indirect Method In case of indirect conjugated method, the primary antibody is
unlabeled. The secondary antibody is conjugated and is directed against the primary
antibody. The antigen-primary antibody-secondary antibody complex is visualized by a
suitable chromogen.
Advantages:
 A single conjugated secondary antibody can be used against different primary
antibodies.
 Higher dilution of primary antibody can be used.
 Large amount of secondary antibody can be easily produced against the primary
antibody.
 For negative control, the primary antibody can be omitted.

Peroxidase-Antiperoxidase Method
Peroxidase-antiperoxidase reagent is an immune complex substance. It consists of horseradish
peroxidase antigen and antibody against horseradish peroxidase. Here a secondary bridging
antibody is used between the primary antibody and peroxidase-antiperoxidase complex. The
primary antibody and peroxidase-antiperoxidase reagents are from the same species,
whereas the secondary linking antibody is from a different species. This secondary
antibody is usually highly specific against the primary antibody and the antibody present
in the peroxidase-antiperoxidase complex.
Advantage:
 High degree of sensitivity: peroxidase-antiperoxidase method is 1000 times more
sensitive than the indirect conjugated method.

Avidin and Biotin Conjugated Procedure

This is the modification of the above-mentioned procedure to further increase the
sensitivity of the test. Here a preformed complex of avidin-biotin and horseradish
peroxidase is used. The presence of multiple molecules of horseradish peroxidase
enhances the visualization reaction.
The steps are such:

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  Primary antibody
 Biotinylated secondary antibody
 Preformed complex of avidin-biotin and horseradish peroxidase
Advantage:
 Highly sensitive
Disadvantage:
 Endogenous biotin may cause false-positive reaction.