Clinical Laboratory Science:

Histology and Cytology

Histology

Specimen Accessioning

Gross Examination

Fixation - Types of Fixatives

The objective of fixation is to preserve cells and tissue constituents in as close a

life-like state as possible and to allow them to undergo further preparative
procedures without change. Fixation arrests autolysis and bacterial
decomposition and stabilizes the cellular and tissue constituents so that they
withstand the subsequent stages of tissue processing. Aside from these
requirements for the production of tissue sections, increasing interest in cell
constituents and the extensive use of immunohistochemistry to augment
histological diagnosis has imposed additional requirements. Fixation should also
provide for the preservation of tissue substances and proteins. Fixation is,
therefore, the first step and the foundation in a sequence of events that
culminates in the final examination of a tissue section.

It is relevant to point out that fixation in itself constitutes a major artefact. The
living cell is fluid or in a semi-fluid state, whereas fixation produces coagulation
of tissue proteins and constituents, a necessary event to prevent their loss or
diffusion during tissue processing; the passage through hypertonic and hypotonic
solutions during tissue processing would otherwise disrupt the cells. For example,
if fresh unfixed tissues were washed for prolonged periods in running water,
severe and irreparable damage and cell lysis would result. In contrast, if the
tissues were first fixed in formalin, subsequent immersion in water is generally
harmless.

A large variety of fixatives is now available, but no single substance or known

combination of substances has the ability to preserve and allow the
demonstration of every tissue component. It is for this reason that some fixatives
have only special and limited applications, and in other instances, a mixture of
two or more reagents is necessary to employ the special properties of each. The
selection of an appropriate fixative is based on considerations such as the
structures and entities to be demonstrated and the effects of short-term and long-
term storage. Each fixative has advantages and disadvantages, some are
restrictive while others are multipurpose. The requirements of a large through-
put diagnostic laboratory are also quite different from those of a research
laboratory with small numbers of specimens for specialised structural analysis
and less requirement for urgency.

Over the years, various classifications of fixatives have been proposed, with
major divisions according to function as coagulants and non-coagulants, or
according to their chemical nature into three general categories which include
alcoholic, aldehydic and heavy metal fixatives. A modification of Hopwood's
classification is shown below:
1. Aldehydes, such as formaldehyde, glutaraldehyde.

2. Oxidizing agents: metallic ions and complexes,

such as osmium tetroxide, chromic acid.

3. Protein-denaturing agents, such as acetic acid,

methyl alcohol (methanol), ethyl alcohol(ethanol).

4. Unknown mechanism, such as mercuric chloride, picric acid.

5. Combined reagents.

6. Microwaves.

7. Miscellaneous: excluded volume fixation, vapour fixation.

Aldehydes include formaldehyde (formalin) and glutaraldehyde. Tissue is fixed

by cross-linkages formed in the proteins, particularly between lysine residues.
This cross-linkage does not harm the structure of proteins greatly, so that
antigenicity is not lost. Therefore, formaldehyde is good for immunoperoxidase
techniques. Formalin penetrates tissue well, but is relatively slow. The standard
solution is 10% neutral buffered formalin (4% formaldehyde). A buffer prevents
acidity that would promote autolysis and cause precipitation of formol-heme
pigment in the tissues.

Glutaraldehyde causes deformation of alpha-helix structure in proteins so is not

good for immunoperoxidase staining. However, it fixes very quickly so is good
for electron microscopy. It penetrates very poorly, but gives best overall
cytoplasmic and nuclear detail. The standard solution is a 2% buffered
glutaraldehyde.
Mercurials fix tissue by an unknown mechanism. They contain mercuric chloride
and include such well-known fixatives as B-5 and Zenker's. These fixatives
penetrate relatively poorly and cause some tissue hardness, but are fast and give
excellent nuclear detail. Their best application is for fixation of hematopoietic
and reticuloendothelial tissues. Since they contain mercury, they must be
disposed of carefully.

Alcohols, including methyl alcohol (methanol) and ethyl alcohol (ethanol), are
protein denaturants and are not used routinely for tissues because they cause too
much brittleness and hardness. However, they are very good for cytologic smears
because they act quickly and give good nuclear detail. Spray cans of alcohol
fixatives are marketed to physicians doing PAP smears, but cheap hairsprays do
just as well.

Oxidizing agents include permanganate fixatives (potassium permanganate),

dichromate fixatives (potassium dichromate), and osmium tetroxide. They cross-
link proteins, but cause extensive denaturation. Some of them have specialized
applications, but are used very infrequently.

Picrates include fixatives with picric acid. Foremost among these is Bouin's
solution. It has an unknown mechanism of action. It does almost as well as
mercurials with nuclear detail but does not cause as much hardness. Picric acid
is an explosion hazard in dry form. As a solution, it stains everything it touches
yellow, including skin.

Tissue Processing

Stabilized tissues must be adequately supported before they can be sectioned for
microscopic examination. While they may be sectioned following a range of
preparatory freezing methods, tissues are more commonly taken through a series
of reagents and finally infiltrated and embedded in a stable medium which when
hard, provides the necessary support for sectioning by microtomy. This
treatment is termed tissue processing. Methods have evolved for a range of
embedding media and applications. Pre-eminent amongst these is the paraffin
wax method, which is considered to be the most suitable for routine preparation,
sectioning, staining and subsequent storage of large numbers of tissue samples.
The quality of structural preservation seen in the final stained and mounted
section is largely determined by the choice of fixative and embedding medium.
During tissue processing, loss of cellular constituents and shrinkage or distortion
should be minimal. After fixation, post-fixation and preparatory procedures, the
four main stages in the paraffin method are dehydration, clearing, infiltration
and embedding.
The first step in processing is dehydration. Water is present in tissues in free and
bound (molecular) forms. Tissues are processed to the embedding medium by
removing some or all of the free water. During this procedure various cellular
components are dissolved by dehydrating fluids. For example, certain lipids are
extracted by anhydrous alcohols, and water soluble proteins are dissolved in the
lower aqueous alcohols.

In the paraffin wax method, following any necessary post fixation treatment,
dehydration from aqueous fixatives is usually initiated in 60%-70% ethanol,
progressing through 90%-95% ethanol, then two or three changes of absolute
ethanol before proceeding to the clearing stage. Duration of dehydration should
be kept to the minimum consistent with the tissues being processed. Tissue blocks
1 mm thick should receive up to 30 minutes in each alcohol, blocks 5 mm thick
require up to 90 minutes or longer in each change. Tissues may be held and
stored indefinitely in 70% ethanol without harm.

Clearing is the transition step between dehydration and infiltration with the
embedding medium. Many dehydrants are immiscible with paraffin wax, and a
solvent (transition solvent, ante medium, or clearant) miscible with both the
dehydrant and the embedding medium is used to facilitate the transition between
dehydration and infiltration steps. Shrinkage occurs when tissues are transferred
from the dehydrant to the transition solvent, and from transition solvent to wax.
In the final stage shrinkage may result from the extraction of fat by the
transition solvent. The term clearing arises because some solvents have high
refractive indices (approaching that of dehydrated fixed tissue protein) and, on
immersion, anhydrous tissues are rendered transparent or clear. This property is
used to ascertain the endpoint and duration of the clearing step. The presence of
opaque areas indicates incomplete dehydration.

Xylene clears rapidly and tissues are rendered transparent, facilitating clearing
endpoint determination. Concerns over the exposure of personnel to xylene
relate mainly to the use of the solvent in coverslipping rather than in processing,
and xylene substitutes can be used in these circumstances.

Infiltration is the saturation of tissue cavities and cells by a supporting substance

which is generally, but not always, the medium in which they are finally
embedded. Tissues are infiltrated by immersion in a substance such as a wax,
which is fluid when hot and solid when cold. Alternatively, tissues can be
infiltrated with a solution of a substance dissolved in a solvent, for example
nitrocellulose in alcohol-ether, which solidifies on evaporation of the solvent to
provide a firm mass suitable for sectioning.
Embedding is the process by which tissues are surrounded by a medium such as
agar, gelatine, or wax which when solidified will provide sufficient external
support during sectioning. Tissues are embedded by placing them in a mould
filled with molten embedding medium which is then allowed to solidify.
Embedding requirements and procedures are essentially the same for all waxes,
and only the technique for paraffin wax is provided here in detail. At the
completion of processing, tissues are held in clean paraffin wax which is free of
solvent and particulate matter.

Sectioning

Frozen Sections

Hematoxylin and Eosin Staining

The H&E combination was proposed shortly after the discovery of eosin in 1871,
although aniline blue was the first counterstain to hematoxylin. Originally, eosin
was used alone to color tissues, but its role now is almost exclusively in double
and multiple staining procedures.

Hematoxylin cannot stain but must be oxidized to hematein (usually at an acid

pH with sodium iodate) which acts as the dye. (Despite this, the staining solution
formed is still traditionally referred to as hematoxylin.) However, even at this
stage except for a few applications, direct staining is unsuccessful and it is
necessary to include a mordant for hematoxylin to stain tissues effectively. The
combination of mordant and dye is known as a 'lake', and in the case of
hematoxylin-mordant, such lakes are often positively charged, behaving as
cationic dyes at low pH. Various metal salts have been used as mordants with
hematoxylin, but only those containing aluminum, iron or tungsten are still in
common use.

Alum hematoxylin solutions have become the standard, universal means of

staining cell nuclei for microscopic examination. Practically every section of
normal and diseased tissue will be examined and presumptively identified using
an alum hematoxylin to color nuclei. The major disadvantage of alum
hematoxylin as a stain is its susceptibility to acids, which limits the range of
counterstains that can be used. Alum hematoxylin staining is also influenced by
other factors, including the concentration and age of staining solutions as well as
the fixation and processing to which the tissue was subject.

The addition of aluminum (at a constant pH), commonly used as a mordant for
hematein, has the effect of increasing the absorbance and hence darkening the
solutions. In this case hematein is indicated by a band at 430 nm, while a 560 nm
peak represents the absorption maximum for the alum hematein complexes.

The most frequently used form of eosin is eosin Y (Cl 45380) which gives
yellowish-pink shades (from the Greek eos, dawn) and can be prepared as either
an alcoholic (2% solubility) or aqueous (40% solubility) solution. Eosin B (Cl
45400), erythrosin B (Cl 45430) and phloxine B (Cl 45410), along with various
similar xanthene dyes, can be used as suitable alternatives.

Eosin Y (M.W. 691.9) is a tetrabrominated derivative of fluorescein with

maximum absorption in water between 515 and 518 nm. Commercial
preparations may also contain fluorescein and tribromofluorescein in sufficient
quantities to influence staining color as the dye becomes more pale with less
bromine. The molecule carries one negative charge between pH 3 and 5 and two
negative charges above pH 5.

Proteins are generally cationic below pH 6 and will thus bind eosin, probably
through the bromine groups. The reaction is influenced by fixation with tissues
prepared in Zenker's fluid, in particular, staining strongly. The selectivity and
strength of eosin staining can also be enhanced by adding a small amount of
glacial acetic to the dye solution.

As a counterstain for hematoxylin, aqueous eosin solutions range between 0.5%

and 2% in strength (1% is most common). Alcoholic formulations generally
contain less eosin. Correctly applied, eosin should stain various tissue structures
shades of pink-especially collagen, cell cytoplasm and erythrocytes. The addition
of a very small amount of phloxine can further improve the result.

Hematoxylin And Eosin Staining Of Paraffin Sections

Most standard tissue fixatives are suitable. Cut sections at 3-4 µm.

REAGENTS REQUIRED

1. Ehrlich's hematoxylin:
hematoxylin (Cl 75290) 6 g
absolute alcohol 300 ml
distilled water 300 ml
glycerol 300 ml
glacial acetic acid 30 ml
ammonium or potassium aluminum sulphate 30 g
sodium iodate 0.9 g
Dissolve the hematoxylin in the alcohol before adding the other ingredients in the
order given. Then mix the solution overnight. The addition of sodium iodate
artificially ripens the hematoxylin so that it may be used immediately.
Alternatively, sodium iodate can be deleted and the mixture ripened by exposure
to warmth and sunlight for approximately two months. The naturally ripened
form has a longer shelf life.

2. Differentiator
1% hydrochloric acid in 70% alcohol

3. Alcoholic eosin solution

95% alcohol 3.9 l
eosin Y (CI 45380) 5 g
phloxine B (CI 45410) 0.5 g
glacial acetic acid 20 ml
4. Ammonia water
0.04% aqueous ammonia

METHOD
1. Dewax and rehydrate sections.
2. Place in hematoxylin solution for 5-10 minutes.
3. Wash sections in running water.
4. Differentiate sections in 1% acid-alcohol and then wash
well in water. Repeat if more
stain needs to be removed. This step requires microscopic
control to ensure that only nuclei are stained.

5.Rinse ('blue') in ammonia water (or similar) for 1 min.

6.Rinse sections briefly in distilled water.
7.Counterstain in eosin for 2-5 minutes.
8.Wash well in water.
9.Dehydrate in ascending alcohol solutions
(50%,70%,80%,95% x 2, 100% x 2).
10. Clear with xylene (3 - 4 x).
11. Mount coverslip onto the labeled glass slide with
Permount or some other suitable organic mounting medium.

RESULTS

Nuclei stain blue; cytoplasm, red blood cells and connective tissue stain shades of
pink.