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1146
Medical Laboratory Technology: Volume 3
10. How much volume of 30% ferric chloride would you need which when diluted with
water will make 100 mL of 5% ferric chloride?
11. What are the steps involved in preparing sections for staining?
12. What is the most commonly used stain for histological sections? What are its advantages
and disadvantages?
13. What is freezing microtomy? State its signicance.
14. How would you maintain a microtome knife for its best performance?
15. What is the use of the following stains and techniques?
(a) Weigert-van Gieson stain
(b) Masson’s trichrome stain
(c) Verhoe s stain
(d) Weigert’s resorcin-fuchsin stain
(e) Periodic acid-Schi stain
(f) Prussian blue stain
(g) von Kossa’s technique
(h) Silver impregnation technique
16. What is the recommended procedure for staining the following?
(a) Reticulum bres
(b) Amyloid
(c) Systemic fungi
17. What do you understand by metachromatic stain? Give example.
18. Make a list of possible problems in cuing sections. How will you resolve them?
Laboratory Techniques in Histology
Venk Mani and Kanai L Mukherjee
Chapter Outline
• Overview
• Logging in of Specimens
• Preparation of Tissues
▪ Fixation
▪ Deformalinization and Secondary Fixation
▪ Decalcication
• Processing of Tissues
▪ Dehydration
▪ Clearing
▪ Inltration (Impregnation)
▪ Manual and Automated Tissue Processing
▪ Embedding
▪ Preparation of Sections
▪ Section Cuing
▪ Preparation of Slide
• Routine Staining Procedure in Histology
▪ Pre-staining Treatments under Special Conditions
37
▪ Preparation for Staining
▪ Staining
• Post Staining Processes
▪ Mounting of Stained Sections
• Special Stains and Staining Techniques
▪ Connective Tissues
• Stains for Particular Substances
▪ Carbohydrates
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1148
▪ Amyloids
▪ Lipids
▪ Pigments and Minerals
• Stains for Microorganisms
▪ General Purpose Stain
▪ Bacteria
▪ Fungi
▪ Tissue Parasite Demonstration
• Staining Kits from Commercial Companies
▪ Special Stains
▪ Special Stains for the Evaluation of Mucins
▪ Use of Special Stains in Commercial Autostainer
▪ Fixative and Mordant
• Frozen Section Technique
▪ Staining of Frozen Sections
• Handling and Embedding Small Tissue Fragments
• Review Questions
Medical Laboratory Technology: Volume 3
Overview
A specimen brought to the histology laboratory must rst be logged, identied and then
subjected to specimen preparation prior to tissue processing. Processed tissues are then cut
into thin sections, stained and mounted for microscopic examination. These steps can be
broadly divided as follows:
1. Preparation of tissues which includes xation and decalcication.
2. Processing of tissues which includes dehydrating, clearing and embedding.
3. Preparation of sections which includes the processes of microtomy, aaching section to
slides (adhesion) and removal of pigments and precipitates.
4. Staining and mounting procedures: dewaxing, staining, dehydrating, clearing and
mounting.
In the following sections, we will discuss the techniques involved in processing the
specimens until they are ready for microscopic examination.
LOgging in Of SpecimenS
The routine histology laboratory receives specimens in the form of biopsies or whole
organs. The strictest aention must be paid to specimen identication. Tragedy may strike
if the specimens are interchanged in the laboratory. For example, a normal patient may be
diagnosed as having the disease, say cancer, and treated for a disease that he does not have,
whereas, the diseased person may not be treated and may die from the disease.
In most hospitals, small specimens are placed in xative solution by the operating room
personnel following biopsy. This protects the specimen from drying. Large surgical specimens
may arrive unxed in the laboratory but they must be put in plastic bags or wrapped in saline
moistened towels and should be kept in the refrigerator until examined by the pathologist.
Laboratory Techniques in Histology
This slows down autolysis and the plastic bag with the saline moistened towel protects the
specimen from drying. If the specimens are mailed outside, they must always be xed prior
to mailing and larger specimens should be opened so that the xative can reach inside. It is
best if the xation of large specimens is done under the direction of a pathologist, to ensure
signicant tissue is not lost or destroyed.
The laboratory must maintain a log book wherein each specimen is entered when rst
received in the laboratory. From the log book the specimen receives an identication number.
Usually the numbers are given as the specimens arrive rather than grouping them. The
interposing of dissimilar specimens helps to avoid possible errors between similar specimens.
The pathology assistant rst examines the specimens. The morphological description of
the xed tissue is narrated by the pathologist and typed by the secretary. This becomes the
permanent record of the patient, much as the hospital chart. Following the gross external and
internal examination of the tissue, a portion of the tissue is trimmed (called a block) by the
pathologist and given to the histotechnician for laboratory processing. It is important that one
of the technicians receive the block so that any special problems of orientation of specimens
and the need for special stains can be discussed.
Aer receiving the block, the technician copies the identication number (same as entered
in the log book), with a so lead pencil on a tag that is always kept aached to the specimen.
The technician now enters a record of the specimen’s identication number into a laboratory
register and the locations of the tissue block taken. In addition, a sketch of the large specimen
is drawn in a separate column on the laboratory register that provides a general outline of the
tissue as it looks.
The specimens with identication tags are then transferred into small plastic cassees with
fresh xative or directly into the cassees as described later. When using an automatic tissue
processor, further processing begins in the late aernoon. If the tissues are in containers, they
are rst taken into perforated cassees, properly wrapped with the identication tag. Several
slices from the same specimen may be placed in the same cassees. These cassees then go
through the subsequent steps of tissue processing prior to microtomy. In the case of bone and
calcied tissues, specimens should be cut into small blocks with a saw before xation. These
will need decalcication before they are lined up with the so tissues for dehydration and
embedding.
1149
preparatiOn Of tiSSueS
Fixation
Fixation helps to prepare specimens for microscopic examination. It is a crucial step. The goal
of xation is to preserve cells and tissues to prevent decay. They should be in a natural “lifelike” state at the time of examination. This is accomplished by stopping the enzyme activity,
killing microorganisms, and hardening the specimens while maintaining sucient of the
molecular structure to enable the subsequent steps of staining successfully. Formaldehyde
(formalin) is the most popular xing agent usually buered by phosphate. The specimens are
ideally xed by immersion in formaldehyde for 6–12 h before they are processed. This will be
further elaborated in following pages.
As mentioned earlier xation of tissues (Figure 37.1) is necessary to prevent subsequent
changes (autolysis). When the tissue is dropped in a xative it is immediately killed and
enzymatic digestive processes are inhibited thus preserving the normal structure. Hence, this
must be done promptly. The other purpose of xing the tissues is to harden them. This should
be done to such a degree that the tissue components and architecture will be lile aected by
any subsequent procedures.
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Figure 37.1 Specimen preparation in histology: (a) Following the surgical procedure, specimens are
sent to the histology laboratory in containers with formalin, (b) A portion of the specimen
is removed, and (c-d) transferred to a smaller container for further processing, (e-f) It is
important that the specimen bears the identication tag
Medical Laboratory Technology: Volume 3
The process of xing is carried out twice. First is at the operation theatre as
where a large amount of tissue or organ may be involved. The re-xing is done in the
histology laboratory aer cuing bits or blocks of tissues submied for histological diagnosis.
The blocks should be thin enough (3–5 mm thick) to facilitate penetration of the xing uid
into the tissue within a short time (3–5 h). Ideally, the volume of the xative should be at least
two times the volume of the tissue block. Specimens must be securely held in a holder along
with the identication number. Mini specimens should be wrapped in lens or cigaree paper
or placed in a special ne mesh capsule before immersing in the xative. If these steps are not
taken, the small segments of tissue (biopsy specimens) may migrate through the perforations
in the capsule and be lost in processing.
From the practical point of view, xatives are classied as routine or special. The most
widely used xative is 10% formalin (Note Formaldehyde gas dissolved in water yields
formalin). It is relatively inexpensive, readily available, easy to prepare, compatible with
most stains and penetrates the tissues well. It, however, has an unpleasant vapour which is
irritating to the eyes. It may cause an allergic reaction on the skin and hence one should wear
rubber gloves while handling formalin and work inside a hood while preparing the solution.
Formalin is slightly acidic which interferes in some staining procedures. In such cases,
neutral formalin is preferred. Formalin is neutralized with calcium carbonate. The routine
laboratory should have other xative solutions available for special purposes. A few of them
are Zenker’s uid, Bouin’s uid, Helly’s uid and Carnoy’s uid.
Preparation of fixatives
Fixatives aect the staining reaction. An ideal xative should be rapid in action, permit a
broad range of staining techniques, and be suitable for a wide variety of tissues processed
collectively. Formalin does not necessarily meet all the requirements discussed earlier. Formalin
occasionally creates a problem in some staining reactions. In these cases deformalinization of
the tissue sections prior to staining will usually rectify the problem. Formalin can cause the
appearance of dark spots, but these can be removed. Under special conditions, other xatives
are used. For example, fats will dissolve in xatives with organic solvents (alcohol, acetone)
while water-soluble materials (e.g. carbohydrate) will be preserved.
Formalin (10%) is the most commonly used routine xative. The commercially available
formalin (formaldehyde in solution) has 40% formaldehyde gas dissolved in water. Hence it
is diluted to obtain 10% formalin xative.
Laboratory Techniques in Histology
• 10% formalin
Commercial formalin (full strength) 100 mL
Distilled water 900 mL
• Neutral formalin
10% formalin solution 1000 mL
Add excess amount of calcium carbonate.
10% formalin is most frequently used for wet storage of
tissue. The tissues are preserved for future use.
Aer the tissue is xed with
the primary xative (formalin), the excess xative is removed by washing briey in water
followed by placing in 70% alcohol. Formalin is extracted more rapidly in 70% alcohol than
in unchanged water.
Formalin sometimes produces a ne dark-brown or
black crystalline precipitate (perhaps due to reaction with haemoglobin). This precipitate is
recognized when the sections are examined under the microscope. To remove the precipitate,
rst deparanize the sections to water (xylene-alcohol-water) and then place in the following
solution:
Ammonium hydroxide (58%) 2 mL
Alcohol (70%) 100 mL
Place the section in this solution for about 1 h, then wash thoroughly in tap water to remove
excess ammonia and proceed to stain. This procedure may not be necessary if the formalin is
of good quality.
1151
The is also known as Here the specimen is carefully examined and the
description includes size and appearance, number of pieces and their dimensions. Larger
pieces may have to be cut into representative pieces from appropriate areas. For example, a
surgeon may take multiple samples from the excision margins of a tumour in order to ensure
that the tumour is completely removed. In case of small specimens, one may have to process
the entire specimen without cuing. Formalin should be removed in the next process. This is
done by placing the tissues in cassees which are small perforated baskets and batches will
be loaded onto a tissue processor for processing through wax.
Deformalinization and Secondary Fixation
Occasionally it is desirable to re-x formalin-xed sections in some other xative (e.g., Zenker’s,
Helly’s or Bouin’s) in order to produce a more brilliant stain. The secondary xation is done
in the following way:
• and “take the section to water”. Then treat it with ammonia water
(30 drops of 58% ammonium hydroxide in 100 mL of water) for 1 h followed by washing
in running water.
Note The expression “Take the section to water” in histopathology laboratory refers to
processing a dehydrated tissue (which means that you have removed the water from
the tissue in previous steps) back to water or hydration. This is needed before staining
as the dyes are water soluble, and cannot penetrate wax-lled sections. Therefore, you
have to bring the cut sections to water, reversing the steps that put wax into the tissue.
Wash with xylene, absolute alcohol (95% and 70%), and then a water wash. Aer this
process the sections are ready for staining.
• Following deformalinization, re-x in Zenker’s or Helly’s uid for 1 h and
wash for 15 min in running water. Residual mercuric chloride is then removed by the
procedure described later. This is called mordanting.
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Medical Laboratory Technology: Volume 3
For Bouin’s mordanting, deformalinize and
rex with Bouin’s uid (or in saturated picric
acid) for 1 h. Remove excess xative by washing
in running water for 30 min (Figure 37.2); rinse
with distilled water and proceed for staining.
Zenker’s uid Zenker’s uid is a commonly
used Tissues preserved by
this method stain well with many techniques
provided the tissues are thoroughly washed
in running tap water for several hours aer
xation. The tissues should not exceed 5 mm
thickness because the penetration power of the
xative is poor. A longer time may be required
for Zenker-xed tissues to take the haematoxylin
stain. Many workers prefer this uid for xing
bone marrow aspirate as the acid present will
decalcify bone spicules that may be in the
specimen. Zenker’s uid is recommended by
many workers for small pieces of tissues, e.g.,
spleen and liver. The xative permits excellent
staining of nuclei and connective tissue bres.
It is recommended particularly for tissues
Figure 37.2 Washing of xed specimen: (a)
Washing for a prolonged period,
(b) Quick washing of section can
be done in a porcelain dish
which are to be stained by one of the trichrome
techniques.
• Stock solution:
Dissolve the following ingredients in 800 mL of water (approximately) by stirring at
room temperature or mild heating. When dissolved, make it to volume (1000 mL).
Mercuric chloride 60 g
Potassium dichromate 25 g
Sodium sulphate 10 g
Distilled water 1000 mL
• Working solution:
Add 5 mL of glacial acetic acid to 95 mL of Zenker’s uid (stock) just before use. The
Hey’s uid Here the stock solution has a lower concentration of mercuric chloride and
potassium dichromate. In addition, the glacial acetic acid is replaced with formaldehyde,
which is added to 95 mL of the mixture at the time of use.
• Stock solution:
Dissolve the ingredients in distilled water (800 mL approximately) by stirring or by mild
heat. When dissolved make to 1000 mL.
Mercuric chloride 5 g
Potassium dichromate 5 g
Sodium sulphate 10 g
Distilled water 1000 mL
• Working solution:
Add 5 mL of formalin (37–40%) to 95 mL of stock solution.
Fixation with either of these chromate xatives (Zenker’s or Helly’s uid) produces greater
cytologic detail than with ordinary formalin. The major disadvantage in the use of these
Laboratory Techniques in Histology
xatives is the necessity of washing overnight in running tap water aer xation in order to
remove the yellow-staining dichromate. In addition, the mercury precipitates in the tissue as
ne granules, which must be removed from the sections before they are stained.
1153
Removal of mercuric chloride deposits
Zenker’s and Helly’s uids contain mercuric chloride which should be removed prior to
staining. The method is described below.
• Deparanize the slide with xylene followed by treatment with absolute alcohol and
95% alcohol.
• Then place the slide in alcoholic iodine solution (0.5% iodine in 80% alcohol) for 5–10 min.
This will remove the deposits.
• Following the iodine treatment, wash in running water (Figure 37.2).
• Then place the section in 5% sodium thiosulphate solution (hypo) for 2–5 min. This will
bleach out the iodine.
• Finally wash the slide in running tap water to remove the hypo and then proceed for
staining.
Bouin’s uid Bouin’s uid is recommended for general purposes and for special study of
haematological and lymphoid tissues. It is made as a single solution rather than stock and
working solutions.
Picric acid, 1.22%
(Saturated aqueous solution) 750 mL
Formalin, full strength
(37–10% formaldehyde) 250 mL
Acetic acid, glacial 50 mL
Fix tissues for 4–18 h. The exact time of xing will depend on the size and density of the
tissue. Following xation wash in several changes of 50% and 70% alcohol for 4–8 h. Residual
picric acid (yellow coloured) interferes in the staining reaction. If a yellow colour is seen in
the section, deparanize, re-wash and re-stain.
Carnoy’s uid Carnoy’s uid is a xative. It is suitable for
cytological specimens and small pieces of tissues (biopsy specimens). It is recommended for
glycogen since aqueous solutions are to be avoided, but it readily removes fats.
Mix the following to make the Carnoy’s uid.
Absolute alcohol 60 mL
Chloroform 30 mL
Acetic acid, glacial 10 mL
Tissues are xed for 1–3 h at room temperature or 12–18 h in the refrigerator (recommended).
Tissues xed in Carnoy’s uid do not require washing. Dehydration of the tissue is already
achieved during xation.
DecaLcificatiOn
When preparing sections of bone, and other calcied tissues, decalcication is necessary in
order to facilitate cuing. Only under special circumstances are undecalcied bones requested.
In diagnostic pathology, most evaluations are made on decalcied sections. Undecalcied
bone sections are examined primarily for the diagnosis of metabolic bone disease.
The calcied hard tissues are rst minimized in size by tearing o the surrounding tissues.
So they are cut into small pieces of approximately 2–6 mm slices with a thin blade hacksaw or
sharp knife. They are then xed in neutral or buered formalin. Tissues must be thoroughly
xed unless a decalcifying agent is combined with the xative. In the following step, the
1154
Medical Laboratory Technology: Volume 3
tissues are thoroughly washed before it is submied for decalcication. Thorough removal of
the xative by repeated washing is important. If this is not done, tissue morphology may be
aected. There are basically only two methods of decalcication:
• Acid method
• Chelating method
Decalcifying reagents are commercially available.
Acidmethod of decalcication Acid method of decalcication is most widely used. The
acid present in the decalcifying uid removes the calcium salt present in the tissue (which
gives the tissue its hardness), thereby renders the tissue so enough for sectioning. The acids
are used as simple dilute aqueous solutions or mixed with other chemicals (xatives).
Out of the many acids, the best results are probably obtained with nitric and formic acids.
• Nitric acid (5%, v/v, aqueous): Add few milligrams of urea to each 100 mL.
Dilute nitric acid solution is probably used most oen as it decalcies rapidly and is
reliable in that it will complete the decalcication. Because of its rapid action, nitric acid
is ideal for urgent bone biopsies. Unfortunately, if tissue is le too long in nitric acid,
considerable damage to the tissue will occur; this damage will become apparent aer
2 days or if in concentrations of acid above 8%. Aer decalcication the tissue is washed
well in 70% alcohol before being processed to paran wax.
• Formic acid (10%, v/v, aqueous)
Dilute formic acid solution is a good routine decalcifying agent; it is slower in its action
than nitric acid, but causes less damage to the tissue by over-exposure. Decalcication
for a piece of cancellous bone 4 mm thick should be complete in 48 h, while for dense
bone two weeks or longer may be required. It is recommended that the formic acid is
changed every two days. The staining results obtained aer formic acid is superior to
those using nitric acid.
With simple acid methods, calcium ions will migrate out of the tissue into the surrounding
solution and make it saturated with calcium. This forms a barrier to further decalcication.
Hence, it is recommended to change the solution frequently. Agitation may also be benecial.
Some workers recommend vacuum at the initial stage of decalcication. It will aid in
inltrating the specimen with decalcifying uid and will draw o carbon dioxide bubbles
that form on specimen surfaces. Some workers suggest suspending the specimens in an
embedding bag. This will expose all surfaces of the specimens to the action of decalcifying
uid, and will allow any precipitated calcium salts to sink to the boom of the container. No
aempt should be made to speed up decalcication through heat or acid treatment. These
may increase decalcication but it will also increase the eects of decalcifying uids on other
tissue components and swelling and maceration will most likely result.
Chelating methodof decalcication Chelating agents (chelate in Greek language means
grab) are organic compounds that have the property of binding certain metals like calcium,
lead, mercury, cadmium, etc. EDTA (ethylenediaminetetraacetic acid) is a chelating agent
oen used in conventional therapy to remove heavy metals (like mercury, lead and others) in
circulation. EDTA, a chelating agent, is a synthetic amino acid that binds calcium. Chelating
method has been applied in vitro (laboratory) for decalcication. It binds the calcium ions and
thus helps in decalcication. Satisfactory results are obtained if EDTA is used in a solution
with pH between 5.0 and 7.2, but a slightly acidic pH (below 7.0) is preferred.
• Ethylenediaminetetraacetic acid (EDTA, 15%, w/v, aqueous), pH 7.2
Laboratory Techniques in Histology
1155
1. Immerse the representative portion of bone in decalcication solution, twice its volume,
for 24–72 h (or longer) in order to remove the calcium out of the tissue. This makes the
tissue so enough for sectioning.
2. The tissue may be mechanically tested for adequate decalcication. Bending or piercing
with a very sharp needle is oen adopted by an experienced histotechnician, but these
procedures may damage the cells and ruin the specimen for histological examination if
done too roughly.
3. Wash the decalcied specimen in running water for 24—48 h. Every trace of decalcifying
solution must be removed before dehydration and embedding. Some laboratories
neutralize the decalcifying acid by treating the block with 10% formalin to which an
excess of calcium or magnesium carbonate has been added. The tissue is now ready for
dehydration, clearing and embedding.
Lile damage occurs to the tissue although it may be in EDTA for several months before
decalcication is complete. The speed with which calcium is removed can be accelerated
by heating between 37–42°C, without causing tissue damage. Staining is of an acceptable
standard aer using this chelating agent and good dierential results can be obtained.
Combination method of decalcication This combination method, using both acid and
chelating agent, is recommended by many laboratories that helps to expedite the decalcication process.
Reagent
EDTA 700 mg
Potassium sodium tartrate 8 mg
Sodium tartrate 140 mg
Conc. hydrochloric acid 99.2 mL
Distilled water (q.s.) 1000 mL
Follow the standard procedure of decalcication described before.
Note The tissue should be le in this solution for no longer than 36 h (one and a half days).
Detectingtheendofdecalcication It is important that when the
tissue should be removed from the decalcifying uid immediately. Both, under decalcication
and over decalcication, create problems in tissue processing:
• Under decalcied tissues will be hard to section.
• Over calcied tissues will have problem in staining.
Howtondtheendpointofdecalcication?–There are three ways to determine the end of
decalcication:
• Mechanicalmethodsinvolve testing the exibility
of the specimen, probing the specimen with a needle or pin, and scraping the section
surfaces. They are least desirable for determining the end point because of their
inaccuracy and the likelihood of creating histological artefacts.
• Chemical methods: These depend on the precipitation of calcium oxalate when a
sample of the used decalcifying uid is mixed with a solution containing ammonium
hydroxide and ammonium oxalate. Approximately 5 mL of the used decalcifying uid
is made neutral to litmus paper with concentrated ammonium hydroxide, and then