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J Clin Pathol 1984;37:1409-1415
Technical methods
Control of rapid nitric acid mineral acids for decalcification but the control at
decalcification intervals of 10-20 min is difficult to achieve accu-
rately with the chemical tests and expensive with x
rays. cheap, and accurate method of control-
WHB MAWHINNEY, E RICHARDSON, AJ MALCOLM A simple, is to measure the weight
Department of Histopathology, Royal Victoria ling acid decalcificationas it is decalcified.' The
Infimary, Newcastle upon Tyne change of the tissue is a controlled rapid
endpoint is clear and the result without adverse alt-
Decalcification is commonly employed in most decalcification over a few hours and with minimal
histopathology laboratories for the microscopical eration to staining properties aqueous nitric acid
examination of bone and other calcified tissues. shrinkage using 5% or 10%
Plastic processing without decalcification may pro- (Figs. 1 and 2). Alma7 in their investigation of
duce superior results in terms of eliminating shrink- Verdenius and used weight loss as an indi-
age and for demnonstrating osteoid versus mineral- decalcifying methods salts are removed.
ised matrix'-' but may give poor cytological detail cator of the rate at which calcium weighed
and is a much longer process. The diagnosis of Their technique and that of Richardson6 depend-
non-metabolic diseases of bone such as infection the block in air and could be prone to error dur-
and tumours requires good cellular morphology and ing on the amount offluid allowed to evaporate of
a quick result to allow rapid therapeutic intervention ing weighing. It is surprising that the control
for optimal patient care. In addition, many modern decalcification using the weighing method has not
treatment regimens for bone tumours require a been more widely used in the light of their clear
rapid report on an entire slab of resected bone to documentation of its suitability. tissue in a complex
allow assessment of the benefit or otherwise of Calcium may expect to leave including the
preoperative drug treatment. For these reasons a manner depending on many variablesof calcium in
rapid method ofdecalcification which gives excellent decalcifying agent used, the amount ratio, and,
and reproducible results was devised. the tissue, overall surface area to volume In
Many alternative decalcification regimens have in particular, the block's smallest dimension. the
been proposed,4 but most of them have some addition, the decalcifying agent has to enter it
unsatisfactory characteristics. In an attempt to block as the mineral salts travel to the exterior and
reduce the commonly encountered artefacts of tis- would not be surprising if the formula describing the
sue shrinkage and adverse staining results obtained loss of calcium was rather complicated.8 rate of
with rapid decalcification in strong mineral acids Verdenius and Alma found the to the size
such as nitric acid many laboratories have utilised decalcification to be directly proportional et a19
the much slower decalcification achieved with of the reaction surface, while Kiviranta
formic acid or EDTA. described the ratio for 2 mm bone slices by the for-
Most unsatisfactory results with decalcification mula:
can be attributed to overexposure to the agent used y = 5458x°4'
due to inadequate control procedures. The com- where y is the percentage of calcium extraction from
monly used control procedures using the ammonium the bone slice and x the duration of demineralisation
oxalate chemical test5 and its modifications are accu- in days.
rate in experienced hands6 but are easily abused. Examination of many examples of weight loss
Furthermore, although control usingx rays is simple curves indicates that the loss of calcium follows
and accurate it is expensive in equipment and con- fairly accurately an exponential decay over the
sumables for smaller laboratories and is only realis- major portion of the curve and the weight of calcium
tic for use at 24 h intervals. in the block at any time during decalcification can be
Normal mechanical methods of "testing" using described by the formula:
"bending or probing' cause artefact and are not W(t) = W(o)e-xt
considered by any serious bone laboratory for con- where W(t) = weight at time t, W(.) = original
trol purposes. To achieve rapid throughput of weight, and X = factor depending on block and
calcified tissues many laboratories resort to using decalcifying agent.
Towards the end point of decalcification the gra-
Accepted for publication 30 July 1984 dient of the curve becomes progressively less nega-
1409
1410 Technical methods
L.I
e
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Fig. 1 Areasofreactive boneshowingwellnucleatedbone withsomeosteoblasts and
marrowfibrosis. (a) Formic acid'0 (which also shows a solitary large osteoclast). (b)
5%aqueousnitricacid (with twosmallosteoclasts). Haematoxylin andeosin. Original
magnification x 560.
tive and may turn positive. At this time the "real ses and averaged values of A may be applied to suc-
weight" of the block in acid may be zero or negative cessive forecasts. Forecasting the end point accu-
due to the formation ofgas bubbles on the surface of rately would allow planning of subsequent proces-
the block and necessitating the use of an additional sing to suit the laboratory, particularly in cases of
glass weight to hold it under the surface of the acid. urgent biopsy material.
It is a simple task for a microcomputer to read an Method
electronic balance, plot a graph of weight loss, and
calculate the state of calcium extraction from blocks. 1 Suspend tissue to be decalcified from an accurate
It should also be possible to forecast the end point to + 1
with improving accuracy as decalcification progres- balance capable of weighing mg.
methods 1411
Technical
Fig. 2 Edge ofan intramedullary chondroma showing cartilage (C) bone and
haemopoietic marrow. Note the lack ofshrinkage ofcellsfrom the bonesurface.
(a) Formic acid'°. (b) 5% aqueous nitric acid. Haematoxylin and eosin. Original
magnification x 900.
2 Cover the tissue with 100 times its volume of 5% This simple method lends itself to control and
or 10% aqueous nitric acid and obtain the weight forecasting of the end point of decalcification manu-
immediately. ally or using an electronic balance outputting the
3 Weigh the block at intervals of 5 min initially and sample weight to a small microcomputer. As a con-
10 min as the weight change is reduced. trol procedure this technique is of great help for
4 Construct a graph of the weight change of the other rapid decalcification techniques or fluids or
tissue as in Fig. 3. indeed control of any procedure involving change of
5 As the end point of decalcification is approached equilibrium conditions detectable as a weight
the negative gradient of the graph decreases and in change. linked to an KB23 elec-
most cases turns positive. AnAcorn Atom Oertling
methods
1412 Technical
0271 Fig. 3 Manually drawn weight loss chart of15 x 6 X 3
0261 mmblockofvertebrae decalcified in 5% aqueous nitric
- 025- . acid.
* .
0 24
.
0-231 .
0 *
022
* .
l o 40 50 60 70 80 90 100 110 120 130
O0 3'0o Tbne (min) Fig. 4 Computerdrawn weightlosschartofa 45 x 21 x 5
mmblockofrib decalcified in 10% aqueous nitric acid.
Total tme 6 h 50 min, intervals 5 min.
. IM-1 IKPM--=Ml;2!v -10.
.. -aa--aa- . . . . . . . . . . . . . L- -
;-.dd . . . . .---. . . . . . . ; GXMD.. ; . . . . . . . . . . . . . . . . . . . . . . .... ;g;.;
II.
LLI N
I;-
(Y)
00
lll
m
af)
z
llJ
c:
u iTHIEi Hi......iii i i i i i i H E E...if
4.............A4.... ....HH H H H. H i i i...i.ift! i i i i
tronic balance has been in use in this laboratory
since January 1984, and although the end point
forecasting subroutine is not yet as accurate as is
desired the combination has been of considerable
help. Set up as in stages 1 and 2 above the computer
programme will take the readings at 5 min intervals,
construct the graph, forecast the end point, and sug-
gest to the user when the end point is reached (Fig.
4). A BASIC programme, of which the flowchart is
shown in Fig. 5, is easily written or copies suitable PRINT
for use on an Acorn Atom may be obtained on disc HARDCOPY
or cassette for £3-50. A fully documented listing of
the programme and details of the interconnecting
cable between computer and balance are available
from the authors on receipt of a stamped addressed
envelope.
Fig. 5 Flowchartofbasicprogramme tomeasureandplot
sample weight change.
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