of 7
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3
ec lcific tion nd
other tre tments for
h rd
t ssu s
Decalcificatien
by
acids
45
Decalcification by cheloting agent 46
0eCIIlciticlltioD in praclicll
41
AciII
dllC8lcifien .
3.3.2
Ole\alio,
Mm EDrA
49
3.3.3. Erld poIm
de :alc,fcation 49
3.4.
Softenilg of
nl)ll-
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WlII
be
dnven from
left to right, removing
the hydroxide ions liberated as a result of
dissolution of
the
hydroxyapatite.
Any
strong acid could
serve
as a source of hydro
gen
ions,
but
Ihose that
form
sparingly soluble calcium
salts
e.g.
sulphuric
acid
are,
far
obvious reasons, unsuitable. Hydrochloric,
nitric and
formic
acids are Ihe
ooes
most often
used.
The
overall
readions of
lhese
adds
with hydroxyapatile
are
respectively:
Ga
1O
P
4
)6 OH)2 + 20H+ + 2 cr
10Ca
2
+ +
2 Cr
+
6H
3
P
4
+
2H
2
0
The
calcium from
the
tissue
ends
up
as
calcium ions dissolved
In
the
decalcifying
fluid. If the
latter
is changed frequently the
completion
of decalcification can be rec
ognized when extraded calcium ions are no longer
delectable
by a
simple
cnemi
calles . .
The aystallanice of the hydroxyapatile of bones
and
teeth
inrorporates
small num
bers of carbonate ions in addition
to the
more
abundant
phosphates and hydrox
ides.
The mineralized
tissue contains, in
effect.
a
small percentage
af
calcium
carbonate.
This is
dissolved
by acids:
Minute bubbles
carbon dioxide
are
fQ lTled
within and on Ihe surfaces
of
speci
mens being decalcified
in adds,
but they do not usually
produce
signs of damage
visible
under
the microscope.
Mast enzymes
are
put out of aaion
by
acid decalcifying
agents,
but the strudure
of the tissue is only slightly disrupted provided that
fixation
has
been adequate.
Immunohistochemical
stilining
is
often
poor after decalcification ilt km p
and
weilker
acids such
as
acetic Henzen-Longmans
al
1965)
or ascorbic,
pH
aboul
2.5, Merchan-Perez
al 1999)
are
preferred
for
most antigens.
Decalcification by
ilcids can
result in
hydro/ysis
of
nudeic adds. RNA is
broken into
soluble
fragments,
V\lth resulting redudion
or 55
of cytoplasmic
staining
by cation
ic
dyes. The
purine and pyrimidine
bases
are
removed from
he sugar-phosphilte
backbone of
ONA; this occurs
less completely
than
in
a deliberille Feulgen hydro/y
sis
Chilpter 9 , bul
nevertheless
inlerferes with
the interpretation of
densitometric
measurements of
histochemically stained sections Of with s tu hybridiziltion
A1es
al 1999 .
In
a
comparison of decalcifying
solutions Shibata al 2000) found
that hybridization
of
labelled probes
with
mRNA
in
dental
tissues
was
severely
impaired by
mineral
acids but was satisfac:l.ory alter
soIutions
containing formic aeid.
by
chelating agenls
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Decalcification
in pradice l. J
Ethylenediamine tetraacetic acid EDTA forms ordinary salts four are possible)
with sodium and other alkali metals, but the ethylenediamine tetraacetate ions
combine with most other metal ions to form stable, soluble chelates.
If
a piece of
calcified tissue
is
immersed
in
liquid containing
EDTA
anions, free calcium ions will
be removed from the solution by chelation. Hydroxyapatite will therefore dissolve
because it will be unable to attain equilibrium with a saturated solution.
[EDTA]2 -
As in a solulion of lhe
disodium sall of EDTA
The bonds to lhe
Ca alom are of
equal lenglh and mutually at
right angles, directed as if to the
vertices of a regular octahedron.
For
a full account of the chemistry of chelation, see Chaberek and Martell 1959).
lhe
chelation of metal ions by dye molecules
is
described in Chapter
5.
of this
book. Decalcification
by EDTA
differs from decalciflcation
by
acids
in
that hydrogen
ions play no part
in
the chemical reaction involved.
lhe
chelating agent
is
used on
the alkaline side of neutrality, so
the deleterious effects of acids on labile sub
stances such as nucleic acids and enzymes are avoided. Strongly alkaline solutions
are not used, however, because they extract proteoglycans from the extracellular
matrix of the tissue lppolito
al
1981). The main disadvantage of EDTA is that
it acts more slowly than the acids.
Note that EDTA has many names, including versene, sequestrene, edetic acid, eth
ylene-bis iminodiacetic acid) and ethyjenedinitrilo)tetraacetic acid, with correspon
ding names for the socIium
salts.
In a solution of any of the salts the number of
ionized carboxyl groups available for chelation increases with the pH. A
1
solu
tion of
Na2EDTA has
a pH of 5.3 whereas with
Na3EDTA
the pH is 9.3 and with
Na
4
EDTA it
is
11.3.
Decalcification in practice
Specimens \hat are to
be
decalcified must
be
properly fixed and no longer respon
sive to changes in osmotic pressure. Usually the flXative must be thoroughly
washed out of the tissue with water prior to decalcification in order to avoid unde
sirable chemical reactions.
For
example, residual sodium phosphates from a
buffered formaldehyde flXative would oppose the action of
an
acid decalcifier.
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l I Chapter 3 Iecalcficaton and other treabnents for hard tssues
300 mi
50
g
6 70 mi
30m
3 3 1
dd decaldfiers
every 3-5 days. If t he ammonlum oxalate test Section 3.3.3)
is
t o b e used, the
anticipated last change of decalcfer should have only about five times the volume
of the specirnen,
so
that any calcum in the liquid wil l be present at higher concen
tration than in a larger volume.
Teeth present various difficulties not encountered with bone. The enamel
consists
almost entirely of calcium salts, with a tenuous framework of protein that usually
collapses after decalcification. The fixative should be one that strongjy
cross-links
protein molecules; glutaraldehyde is suitable.
lh e
pulp can shrnk, tearing the odon
tablast processes, which pass from the pulp into the dentine.
lhis
artifact is
also
reduced by glutaraldehyde fixation van Wyk, 1993).
Formic acid, at pH 1.5 t o 3.5
is
the decalcifier of choice for most purposes.
Several
mixtures similar to the one below have been described. Sorne of them also con
tain formaldehyde.
in an
attempt to offset the consequences of inadequate
primary
fixation.
ther
organic acids used for decalcifcation indude acetic and ctric, which
are slow, and lactic, which
is
nearly as fast as formic Eggert and Germain, 1979).
Adequately fixed specimens severaI days in formaldehyde; shorter times in more
rapidly acting fixatives)
can
be decalcified in 1.0 M hydrochloric or nitric acid
C h a p ~ r
20).
lhese
mineral acids pH
=
remove calcified deposits more
quick
Iy than formc acd see Sanderson, 1994). De Castro s fluid is one of many older
decalcifying fluids that contain a mineral acid. Ascorbic acid
is
suitable only for del
icate objects containing only thin layers of bone.
An acid decalcifier typically takes
4-5
days
to
soften a
5
mm
cube
of
cancellous
bone Culling, 974). Nilsson
et
l (1991) perfused mts with a flxative, followed
by an acid decalcifier for
6
h. This was
as
effective
as
immerslng fixed tissues
for
h i n t he sa me liquid. A more generally applicable way to reduce the time con
sists of heating to 55C
in
a microwave oven. lhis
is
said t o p ro du ce a lO-fald
acceleration of t he reaction Kok and Boon, 1 99 2).
lhus,
a pece of bone requir
ing 3 h to decalcify at room temperature can be ready to dehydrate and embed
after
as
little as 3 h in at C
When decalcification is complete Section 3.3.3) the speClmen should be washed
in severa changes of water or 70 alcohol.
uffered formc cd
C1ark, 1954
Formic acid (90 ): 250 mi
Water:
750
mi
Sodum formate HCOONa):
34
g
Alternatively, a solution with the same compositlon
can
be made by
dissolving
19.8g of sodium hydroxide in 729 mi water and adding 271 mi of 9 formic
acid.
This solution keeps ndeflnrtely. Its pH is 2.0, and it produces only minimal suppres
sion of the stainability of nucleic acids. Ippolito
et 1
(1981) found that formic
acid
at pH 2.0 extracted less proteoglycan from cartilage than 5 other decalcifying
agents tested.
De
astro s
fluid
Absolute ethanol:
Chloral hydrate:
Water:
Concentrated nitric acid (70
HN 0
3
Mix in arder stated. Keeps Indefinitely.
Cauton. The nitric acid must be added last. Concentrated HN0
3
reacts explosively
with concentrated ethanol.
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1 0
g
0.85 g
100ml
3 3
heJation with
E T
3 3 3
End point
o
decalcificaton
3.3 Decalcification
in praetice I
De Castro s flUid is traditionally used in conjunction with neurological staining meth
ods. It
is
strongly acidic and decalcifies rapidly. The alcohol and chloral hydrate in
the mixture
are
supposed to prevent swelling of the tissue, presumably by ncreas
ing the osmotic pressure of the fluid. This may be unimportant when adequately
flxed specimens
are
being decalcified. However, de Castro s mixture was formerly
used
as
a simultaneous fixing and decalcifying agent
In
this circumstance the
osmotic effects,
as
well
as
the fixative properties of
al
three ngredents, assumed
greater slgnificance.
scorbc ocid n so ne
(Merchan-Perez
a 1999
Ascorbic acid:
Sodium chloride:
Water:
This solution is prepared immediately before use and is used only once. It is suit
able for small specimens, notably the inner ears
of
small animals, with which
Mercha
n-Perez
a/.
1
999) found immunohlstochemical
sta
i
ni ng
for various antl
gens to be superior to that seen after decalcfication in
EDTA.
Decalcification wlth EDTA proceeds much more slowly than wth acids, several
weeks occasiona 1y being required, but this
is
not Injurious to the t;ssues.
EDTA
s
frequently used when the sectlons are to be stained with immunohistochemical
methods, and
s
able to unmask some antgens (Hosoya a/. 2005; see also
Chapter 19). After decalciflcation with
EDTA
some histochemical methods for
enzymes can subsequently be performed upon frozen sedions.
An
EDTA
solution
is
made up immediately before using and
is
used only once.
Dsodum EDr solution
Thls solution
is
recommended for routine decalciflcation.
Either 5 or 10 g of disodlum ethylenediamine tetraacetate, [CH
2
N(CH
2
COOH)
CH
2
COONah 2H
2
0, is dissolved in 100 mi
of
water, and 4 NaOH is added until
the pH
is
between 7 and
8.
mmonum EDr soluton
This solution is recommended for specimens containing dense bone. Sanderson
al. 1995) found that t took 6 days to decalcify material that required 18 days in
other
EDTA
mxtures.
Ethylenediamine tetraacetc acid [CH
2
N(CH
2
COOH)2b: 14 g
(Note that this is
EDTA
acid, not one of its salts.)
Ammonium hydroxlde (ammonla solution, 28-30 SG
0 9 :
9 mi
Water:
76
mi
Stir until dissolved, then
add
more ammonium hydroxide until the
pH is 7 1.
The specimen is put in a perforated plasbc casette in the jar contaning the EDTA
solution, and left on a magnetic stirrer to keep the liquid
In
gentle motion while
decalcificatlon proceeds.
An EDTA
soluton should be changed every third or fourth
day. when deca cified, the specimens should be washed in water befare dehydra
tion, because the
EDTA
salts are insoluble
In
alcohol. (Alcohol-soluble
salts
of
EDTA
are avallable, but accordi ng to Eggert
et
o 1981) they have no special
adva
ntages
as
histological decalcifying agents.)
If
the specimen
is
large enough, extends beyand the reglan of interesl and con
tains calcified tissue throughout,
it
may be trimmed with a razor blade or scalpel at
intervals during decalciflcatian. When it can be cut
easlly,
it will also be 50ft enough
to be sectioned
on
a microtome. Sometimes the specimen
is
too small to be
trimmed, ar the calcifled material is isolated
In
the middle of the block. An
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l 50 Chapter
3
I
Decaicification and other
treahnents
for hard tissues
othelWise unwanted piece of bone of approximately the same size can then be
processed alongside the specimen. When this piece of bone is fully softened the
specimen for hlstological study should also be decalcified. Specimeos should never
be tested by poking needles into them; the holes will persist in the sectons.
A more
exaet
test for completeness of decalciflcation makes use of the facl that cal-
cium oxalate, though soluble in mineral acids, is insoluble in water aod io aqueous
solutions of alkahs.
CaZ
+
+
e o
2
+
HZO
-
Z 4
The lest is conducted as follows:
1)
Add drops of strong ammonia solulion (ammonium hydroxlde, se 0.9 to
about 5 mi of used decalcifying fluid until the mixtu re becomes alkallneto lit-
mus paper (pH > 7).
2 Add 5 mi of a saturated aqueous solution of ammonium oxalate (approxi
mately 3 (NH4)2C204.H20; can be kept
00
the shelf for years) and leave
to stand for
30
min.
l no white precipitate
has
formed after this time, the fluid contains no calcium ions.
Thls test can also be used
to
determine the end-point of decalcification by
EDTA
even though solutions
of
the latter do not contain free calCium ions (Eggert
and
Germain, 1979). lh e [caEDTAF- anion presumably dissociates as the highly insol
uble oxalate is formed. Rosen
1
981 recommends adjusting EDTA solutlons to pH
3.2 3.6
for maximum sensitivity of the oxalate test.
If
an
X ray machne is available,
me
presence
or
absence
of
calcified deposits in a
speClmen can be demonstrated by radiography. Control specimens known to con
talO and not to contain calcified materiaI should be X .rayed alongside the speClmen
being testE;d. in order to assess the amount of radio-opacity due to soft tissues.
It.is
p ~ ~ l a r
i m p o r t ~ t t? determine the e n d p o i ~ t
of
decalcificaton when 1
0
M
n1tnc or hydrochlonc aCld IS used, because excesslve exposure to these solullOns
will cause hydrolysis of proteins and macromolecular carbohydrates
as
well
as
nudeic acids, with consequent struetural damage.
3.4.
oftening
non calcareaus materials
Cartlage
in
vertebrates and chtin
in
arthropods are composed largely of macro
molecular carbohydrates, but they often also contain insoluble calcium salts.
lhese
materials can be softened to some extent by demneralizaton in acids or chelating
agents. The hard external layer
of insed
cuticle can be softened with
an
alkahne
hypochlorite solution and removed by careful dissecton (Haas, 1992 .
Wood
is
cellulose, reinforced by lignin (Chapter 10), and aften contains deposits
of silica Si0
2
Crystals of silica
can
cause sectioning difficultes with other
tissues
including leaves of some grasses. Hydrofluoric acid HF , whlch dissolves silica
,
is
sometimes used as a softeoing agent for hard plant tissues. The concentration
may
range from
15
to 60 depending on the hardness, applied for
12 36
h (see
Ruzin 1999). If calcium silicate is also present, the solution should also include a
little sulphuric
aCld
(Tomasi and Rovasio 1997).
xtreme c uton
is needed when
using concentrated hydrofluoric acid: protectlon of the eyes hands (nitrile or natu
ral
rubber gloves) and body (natural rubber or neoprene apron) and an adequate
7/25/2019 Capitulo Descalcificacion
7/7
3.5 Softening specimens
already embedded in
wax I
fume hood. Calcium carbonate or hydroxide powder should be to hand for neutral
izing spills. Calcium fluoride is insoluble and not hazardous. Many institutions
require specific training of personnel who are to use HE
Carlqust
1982)
preferred a solution containing ethylenediamine.
Its
mechanism
of adion
is
uncertain. The original author used 4 ethylenediamine, prior to desili
cifying very hard woods with 24
HF Kul
