Decalcification

Bone decalcification is the removal of calcium ions from the bone

through histological process thereby making the bone flexible and easy for
pathological investigation. This is necessary in order to obtain soft sections of the
bone using the microtome. Every thin section cut can be processed like any other
soft tissue of the body. Calcium ions found in the bones are responsible for its rigid
posture, people suffering from diseases like osteomalacia and rickets have
unusually low amounts of calcium ions in their bones thereby rendering their bones
flexible and most times unable to carry their body weight. There are two categories
of decalcifying agents namely, chelating agents and acids. The acids are further
divided into weak (picric, acetic and formic acid) and strong acids (nitric and
hydrochloric acid). The acids make up a solution of calcium ions while the chelating
agents take up the calcium ions. Most frequently used chelating agent is
Ethylenediaminetetraacetic acid (EDTA). Acids have some effects on the
stainability of the tissue.
Decalcification is a lengthy procedure, as bone pieces have to be left in the
decalcifying agent for several days or even weeks, depending on the size of the
bone. Traditional methods of handling hard tissues, i.e. bone and teeth, usually
present a problem to both the pathologist and histotechnologist. Many of the
grossing and cutting-in techniques in current usage for these tissues dictate the
use of gross sectioning procedures with a high-speed saw and/or long periods in a
decalcifying solution, prior to reducing the specimen to a size that can be easily
processed, embedded and sectioned. Frequently the poor quality thin sections
obtained when these methods are employed contribute to the already difficult task
of evaluating the pathology and making a correct diagnosis. With the introduction
of Low Speed Saws, it is now possible to routinely and rapidly reduce undecalcified
surgical specimens of hard tissue, to a thickness of 23 mm, without compromising
the integrity of the tissue.
There are numerous tests which can be done to ensure that decalcification is
complete. These tests are physical which involves simply bending the specimen, xray examination and chemical means.

RESIN PROCESSING OF BONE

FIXATION
1. The specimen is allowed sufficient time for it to be fully fixed with 10% formol
calcium (at least 24 hours after removal from the body) before processing is
commenced. Specimens which are labelled with tetracycline (noted on request
form) for subsequent demonstration with fluorescence, must be fixed only in 70%
alcohol and not formol calcium, for at least 24 hours after removal from the body.
2. If the specimen is too large, it should be trimmed/cut so that adequate size
block(s) are taken from the part(s) required for examination. Surplus tissue not
required for examination should be cut off. Should cutting/trimming be necessary,
then advice must be sought from an experienced MLSO.
3. Large specimens especially those which require cutting/trimming may require
longer fixation than 24 hours before processing is commenced.

PROCESSING
The following processing schedule is suitable for biopsy size specimens which are
processed in a 10ml glass vial.
1. 70% Alcohol 2 hours 2. 90% Alcohol 2 hours 3. 100% Ethanol x 2 2 hours each 4.
Ethanol/methyl methacrylate monomer (1:1) overnight 5. Infiltrating solution A x 2
4 hours each 6. Infiltrating/embedding solution B 23 hours
NOTES
1. Specimens must only be processed under an operational fume hood.
2. Specimens are agitated continuously on a roller mixer for all steps.
3. Waste solutions containing alcohol can be discarded down the sink.

4. Any waste solution containing methyl methacrylate must be discarded after

being allowed to evaporate off according to waste-dosposal policies.
INFILTRATING SOLUTION A (STORE AT 4C)
--Methyl methacrylate monomer (stabilised) 70ml
--Dibutyl phthalate 30ml
--Benzoyl peroxide (previously dried) 2g
Make up solution under a fume hood in a darkened bottle. Benzoyl peroxide must
be dissolved before use by agitating on a roller mixer.
INFILTRATING/EMBEDDING OF SOLUTION B (STORE AT 4C)
--Infiltrating solution A 100ml
--Poly methyl methacrylatelow mol wt beads 30g
Make up mixture under a fume hood in a darkened bottle, mixing on a roller mixer
until beads have fully dissolved (approximately 4 days)
DRYING BENZOYL PEROXIDE
Benzoyl peroxide is supplied damped with water as it is potentially explosive in its
dry form. However, it is essential that only dried benzoyl peroxide is used in the
solutions A and B. It is dried as follows:
1. Spoon out approximately 10g of benzoyl peroxide into a filter paper bent in the
shape of a cone on a beaker.
2. Dry in a 37C oven overnight. Use only the oven in the laboratory, and place a
sign to warn staff. 3. Excess dried benzoyl peroxide is stored on the shelf in the
container provided away from direct heat.

EMBEDDING
1. Allow specimen in resin embedding solution B to stand for 1 hour.
2. Orientate the label using a wooden cocktail stick, so that it is bent at right
angles across the specimen.
3. Secure lid on vial/container.

4. Partially immerse in pre-heated 60C water inside a staining dish. Ensure lid is
replaced on dish.
5. Polymerise resin overnight at 60C in the oven. Use only the 60C oven in the
resin laboratory.
RELEASING BLOCK FROM GLASS CONTAINER
1. Place glass container (minus lid if possible) in freezing compartment of the
refrigerator for 15 minutes.
2. Wrapping the container well in paper towels, break the container by hitting
gently with the hammer provided. Wear eye protection.
3. Discard both waste paper and broken glass in the glass bin, and ensure no glass
is lying on the bench.
4. Carefully wash block(s) under a running tap.

SECTIONING
1. Clamp block in specially designed holder on Reichert-Jung Autocut so that the
bone is vertical to the knife edge.
2. Using the trimming tungsten carbide knife, trim into block until a representative
area is reached, lubricating both knife and block with 30% alcohol with the aid of
an artist's brush.
3. Remove the trimming knife and replace it with the cutting tungsten carbide
knife and cut sections at 6, lubricating both block and knife as previously
described. Sections that require fluorescence to examine tetracycline labelling
should be cut at 12 and kept separate from other sections. Sections can be picked
up using a pair of fine forceps.
4. Cut at least 6 sections per routine case so that spares are available should they
be required.
5. Store sections in 30% alcohol (or 70% alcohol for tetracycline-labelled tissue).
Use a separate labelled small specimen pot for sections from each block.

PROCESSING OF EYE

Indications
Eyes may be removed because of intraocular tumors (retinoblastoma in children
and melanomas in adults), to relieve pain in eyes that have become blind, and
eyes which have become infected. It is possible for patients to function well after
the surgical removal of an eye, and an excellent cosmetic result may be achieved.
Eyes may also be removed as part of the postmortem examination. Insights from
the examinations of such eyes have advanced vision research greatly. Patients who
wish to donate eyes for research should contact their local eye bank for
information. Patients may wish to contact their local Eye Bank. In some cases,
especially following the death of infants and very young children, the eyes may
become an important part of the forensic investigation into the cause of death.
Preparation by Ophthalmologist
It is not necessary for the ophthalmologist to open the eye to improve fixation, nor
is it desirable for the ophthalmologist to inject any fixatives into the eye. Such
maneuvers may produce artificial retinal detachments.
The eye should be submerged in a suitable fixative (e.g., 10% neutral buffered
formalin). If the eye is to be sent to a specialized eye pathology laboratory such as
ours, it is important to ensure that the eye is completely submerged in fixative and
that the container is sealed properly to avoid leakage during shipping.
Optimally, eyes should fix for at least 48 hours before dissection in the laboratory.
Preparation by Ophthalmic Histotechnologist/Cytotechnologist
The processing of an eye requires specialized training. It is advantageous to use
specialized paraffins and clearing agents in the tissue processor. Proper expansion
of the eye using a double waterbath technique allows for the presentation of a
rounded eye on a histology slide.

PROCESSING OF TESTES
It is essential that the testes are fixed in an appropriate fixative such as Modified
Davidsons or Bouins Fixative. They should NOT be fixed in formalin since these results in
cellular shrinkage and precludes detection of the types of subtle morphological changes
that are likely to occur with endocrine disruption. Modified Davidsons fixative is preferred
to Davidsons fixative since PAS stain does not stain the spermatid acrosome (required for
detailed staging of the spermatogenic cycle) when conventional Davidsons fluid is used.

Bouins fixation results in differential tubular shrinkage, with tubules in the center of the
testis showing greater shrinkage than more peripherally located tubules. The shrunken
central tubules are often surrounded by interstitial proteinaceous fluid. This is an artifact of
fixation and should not be mistaken as a real finding. A transverse or longitudinal section
of the testis may be used, or one of each may be employed. It is important to include the
rete testis in the section since this can provide evidence of estrogen induced fluid
disturbances in the rete and efferents ducts. It is important to avoid trauma and squeezing
of the testis during the necropsy dissection since this will result in sloughing of the germ
cells into the tubular lumen, which may be mistaken for a test article related change.