Scandinavian Journal of Clinical and Laboratory

Investigation

ISSN: 0036-5513 (Print) 1502-7686 (Online) Journal homepage: http://www.tandfonline.com/loi/iclb20

99m
Standardized Sterile Kits for Preparation of Tc-
Labelled Albumin, Macroalbumin, Iron Ascorbic
Acid Complex, and Antimony Sulphide Colloid

P. Bruun

99m
To cite this article: P. Bruun (1971) Standardized Sterile Kits for Preparation of Tc-
Labelled Albumin, Macroalbumin, Iron Ascorbic Acid Complex, and Antimony Sulphide
Colloid, Scandinavian Journal of Clinical and Laboratory Investigation, 28:3, 313-323, DOI:
10.3109/00365517109095706

To link to this article: http://dx.doi.org/10.3109/00365517109095706

Published online: 08 Jul 2009.

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 Standardized Sterile Kits for Preparation of
99mTc-LabelledAlbumin, Macroalbumin, Iron Ascorbic
Acid Complex, and Antimony Sulphide Colloid
P. BRUUN
Division of Isotopes,
X-ray Department,
Vejle sygehus, Vejle, Denmark

Bruun, P. Standardized Sterile Kits for Preparation of QQmTc-Labelled Albu-

min, Macroalbumin, Iron Ascorbic Acid Complex, and Antimony Sulphide
Colloid. Scaizd. J . din. Lab. Invest. 28, 313-323, 1971.
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SsniTc-labelling procedures are described. They are standardized and simple

enough for technicians without special chemical training to perform, so that
smaller hospitals with facilities for isotope scintigraphy can produce their own
9QnlTc-labelledcompounds. The experimental background for the procedures
is described.
Key-words: Scintigraphy; Q9n1T~albumin; QQnlTcantimony sulphide colloid;
99niTc iron ascorbic acid complex; QQm'T~ macroalbumin
P. Bruun, M.Sc., Divisiori of Isotopes, Vejle sygehus, DK-7100 Vejle, Denmark

In recent years an attempt has been made to
replace long-lived P- and y-radiating isotopes
(1311, 19SAu, 85Sr, 203Hg) used in radioisotope
scintigraphy, with short-lived y-radiating iso-
topes such as 9grnTc, *7mSr,113mIn,and Ig7Hg.
I n this way the radioactivity in the patients may
be increased, giving a better picture quality,
while at the same time the patients' radiation
dose is reduced. 9gn1Tc (0.14 MeV y-radiation,
Ti = 6 hours) is suitable for scintigraphy and
particularly for the gamma-camera. 9grnTcis the
daughter product of 99Mo ( T i = 67 hours)
and is eluted as pertechnetate ions (Tcod-)
from a 99M0 column generator, using phys-
iological saline.
For several years 9gmTc as TcOs- ions has
been used very successfully for brain scintig-
raphy. Several labelling methods (1, 4-16, 18)
have been developed for the scintigraphy of
other organs, but the use of these methods
routinely in different laboratories has repeated-
ly given rise to problems. For this reason many
laboratories have used 113mJn, as the labelling
procedure was easier to perform. However the
use of 113nl1n involves other problems; 113mIn
has the short half-life of 1.7 hours and a y-ra-
diation energy of 0.39 MeV, which is less suit-
able for the gamma-camera.
I n the last two years standardized procedures
for 99mTc-labelling have been developed in our
laboratory. All reagents and ion-exchangers are
prepared in the form of sterile kits at a local
pharmaceutical laboratory. Simply by following
the procedures exactly, even technicians with-
out chemical training can label with 99mTc. The
procedures avoid the use of the ordinarily open
ion-exchange resin column, p H meter, etc.
I n the following sections the experimental
basis of the procedures is described. 99*Tc was
eluted from 99Mo generators obtained from
Philips-Duphar ('9emTc stercow').
Q9"ITc-LABELLED ALBUMIN
Labelling of albumin with 9gn1Tc (4, 5, 9, 1 1 ,
12, 13, 14, 16) has for many years been un-
 314 P . Brriun

controllable, and many more or less necessary 99111T~ albumin in the preparation of 99IIITc
steps have been added to the procedures. Pers- macroalbumin. Albumin was the only reagent
son & Liden (12) described a series of experi- which could not be prepared sterile in ampoules.
ments which could help to explain many of the 0.10 ml of a 20 per cent human serum albumin
problems, amongst others the close dependence (for infusion) was withdrawn into a sterile
upon pH. syringe from a solution stored in a refrigerator.
The principle of labelling is as follow: TJ!’lllTc Bacterial control of two bottles, both used for
eluate is added to a suitable solution of ferric several months, showed no bacterial contamina-
chloride and ascorbic acid. When the pH is Con.
raised to 7-9, 99111Tcis reduced from the higher At first when 99111Tc-labelled albumin was
oxidation level and becomes complexed with used to determine the ratios of the counting
iron ascorbic acid. Albumin is added and the rates of the kidneys and the heart for ‘renog-
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pH is lowered to 2.0. This pH is quite critical. raphy with blood background subtraction’
oemTc is bound to albumin by shaking for (2, 3), it was observed that the activity after in-
about 15 minutes. Finally the pH is adjusted to jection increased steadily in the right kidney. As
about 5 and unbound 99*‘ITc is removed by a function of time this activity increased
ion-exchange. asymptotically to a maximum value correspond-
ing to colloid retention in the liver. The expla-
nation of this observation was a partial de-
Studies on the labelling procedure naturation or perhaps just a bialbumin con-
Stabiliiy of the ferric chloride-ascorbic ucid tent in the labelled product. There was no re-
solution. Ferric chloride-ascorbic acid solution tention in the liver when a 20 per cent serum
is oxidized slowly, the reduced ferrous ion albumin from Statens Seruminstitut, Copenha-
slowly becoming ferric ion. Most of the earlier gen, was used. This problem should be borne in
procedures therefore stated that a fresh ferric mind when albumin is purchased for functional
chloride-ascorbic acid solution must be used. analysis, for example renography. It is less im-
Experiments have shown that when a large ex- portant in scintigraphy.
cess of ascorbic acid was used Purification o f 9giiITc albumin. The unpuri-
fied 99ll’Tc albumin will contain two impurities
cascorbic acid
( cFeC1,
=3j of importance, f’olllTc iron complex and
99lrLTc04-.Charamza & BudikovP (4) point out
the yield only fell from 95 per cent to about 80 that many procedures in the literature give an
per cent in 2 months. Instead of sterilization apparently large 98n1Tcalbumin yield because
the solution used is heated at 100 OC for only 0 9 1 1 1 T ~iron complex is not completely re-
20 minutes, because ascorbic wid is not very moved. Chromatographically the complex oc-
stable at higher temperatures. curs together with albumin. For example, the
p H adjustment. When a kit is used, pH ad- two compounds cannot be separated with
justment must be standardized with a suitable Sephadex G-25 (cf. Fig. 2 curve a, and Fig. 3).
degree of accuracy. The critical stage is that A method which can remove both impurities
at pH 2.0. The problem was solved by using must be used. Figs. 1 and 6 show adsorption of
given volumes of 1 molar HCI and NaOH in the two compounds to different anion-
ampoules. The correct volumes were pre-de- exchange resins. Dowex 1-x4 is probably the
termined by titration. With every new batch best for the adsorption ot both 99I1lTc iron
the volumes should in principle be controlled complex and Tc04-.
by titration. I n practice this was shown to be A product suitable for use in the kit was ob-
unnecessary. tained by shaking for 10 min with approx.
Albumin. The yield depends on the amount 2.5 ml Dowex 1-x4 (100-200 mesh). The resin
of albumin (12). 20 mg gave a good yield and is put in a glass tube with a glass filter disc in
was found to be suitable for the eventual use of the bottom and vial diaphragms in both ends

'
I\\\?
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2 5 10 20
min
Fig. 1. gQ1llTcO4-in the supernatant as a function of
shaking time and anion-exchange resins. I g dry
resin shaken with 6 ml 0911lTc eluate.
0 Dowex 1-x2 (50-I00 mesh),
Dowex 1-x2 (100-200 mesh).
Dowex 1-x4 (100-200 mesh),
Amberlite IRA 400 (20-SO mesh),
0 Dowex 1-x8 (100-200 mesh).
+ Dowex 2-x8 (20-50 mesh).
Q Dowex 3 (20-SO mesh).
(obtained from Philips-Duphar). The procedure
is standardized so that the volume to be added
to the resin does not exceed the 5-6 ml which
the tube with its resin can contain.
Determination of the yield and purity. The
TcOd- content before ion-exchange is dependent
o n the age of the FeCly-ascorbic acid and was
found by separation with Sephadex G-25 col-
umn (0.8 X 11 cm) to be 5-15 per cent. The
yield of albumin is determined by measuring
activity before and after ion-exchange (2.5 ml
Dowex 1-x4) and is at least 80 per cent (80-
95 per cent). A blind labelling without albuxin
followed by ion-exchange demonstrated that
the activity in the solution was approx. 5 per
cent. I n the example with a minimum 80 per
cent 99InTc albumin yield, there must be
approx. 5 per cent of (100 -80) per cent equiva-
lent to 1 per cent of 99Il1Tc iron complex and
TcOd- in the solution. This 1 per cent will
Y""lTc-LabellirigProcediires 315
CPS
10
10
10
I
10 I I I I I I
10 m l eluate
Fig. 2. Separation of 69111T~
albumin from CotllTcO
4-
on Sephadex G-25 (0.8 x I 1 cm). Eluant: Physio-
logical saline. a) After purification with Dowex
1-x4. b) galllT~albumin from a) with 2.0 %
sQWc04-added. The hatched area corresponding
to RQ111T~04- was determined to approx. 1.8 7%.
largely be gS1llTc iron complex when a fresh
FeC1:Jascorbic acid solution is used. Fig. 2 ,
curve a, shows a 9S1'*Tcalbumin product after
ion-exchange, and curve b shows the same pro-
duct with 2.0 per cent 9 g t 1 1 T c 0 4 - added. The
latter shows clearly a Tc04- peak (hatched area)
1.8 per cent of total.
To summarize: 991nTc albumin yield is de-
termined by measurement of activity before
and after ion-exchange. The yield is 80-95 per
cent, depending o n the age of FeCls/ascorbic
acid solution. T h e product contains maximum
1 per cent of 99"ITc iron complex and 9 9 1 1 1 T ~ 0 4 - .
As an extra control the final p H must be
approx. 5 (pH paper).
When a kit is used, all manipulations are
carried out under sterile conditions. If there is
any doubt about sterility, the preparation can
be finished with a sterile filtration through a
Millipore filter (0.22 btm).
 316 P . Brrcun
Our 99nlTc labelled albumin has been made
for over a year with varying batches of reagents
without any complications. It is used for: 1) de-
termination of kidney/heart ratios used in
'renography with blood background subtrac-
tion', 2) production of 99111T~ macroalbumin
for lung scintigraphy.
Preparation of reagents
Sterile apparatus and sterile, pyrogen-free,
physiological saline are used. FeC13/ascorbic
acid and NaOH ampoules are stable for 2-3
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months.
A. FeCl3iascorbic acid in vials: 2.70 g FeC13,
6 H 2 0 (p.a.) are dissolved in approx. 75 ml
physiological saline in a 100 ml flask. 5.30 g
ascorbic acid (p.a.) are added and the flask
filled to 100.0 ml. 0.50 ml k 0.01 ml por-
tions are pipetted intG 20 ml vials stop-
pered with a rubber diaphragm. The vials
are heated for 20 minutes at 100 "C.
B. 1 molar NaOH ampoules: 4.0 g dry NaOH
pellets (p.a.) are dissolved in approx. 75 ml
physiological saline in a 100 ml flask and
the flask filled to 100.0 ml. 0.38 ml t- 0.01
ml portions are pipetted into 1 ml ampoules
(losses in the ampoule and syringe are al-
lowed for in the 0.38 ml). The ampoules
are sealed and sterilized.
C. 1 molar HCI ampoules: 8.5 ml conc. HC1
(p.a., 37 per cent, e = 1.19 g/cm3) are
added to approx. 75 ml physiological saline
in a 100 ml flask, and the flask filled to
100.0 ml. 0.58 ml & 0.01 ml portions are
pipetted into 2 ml ampoules (losses in the
ampoule and syringe are allowed for in the
0.58 ml). The ampoules are sealed and
sterilized.
D. Ion-exchange columns. (Minimum volume
10 ml): Approximately 100 g Dowex 1-x4
(100-200 mesh) are added to 0.7 1 physio-
logical saline and washed for 4 hours, using
a magnetic stirrer. The liquid is decanted.
Washing and decanting is repeated 5 times.
2.5 ml ion-exchange resin are added to each
column. Two vial diaphragms are used to
seal each column, and the columns are
sterilized. The preparation of the first 3
reagents (A, B and C) must be co-ordinated,
so that the correct pH intervals are ob-
tained during the actual 9emTc labelling
procedure. Normally this is no problem, but
care must be taken when new batches of
chemicals (especially HCI and NaOH pel-
lets) are used. The following methods are
available for controlling the reagents.
1) Carry out a trial labelling and mea-
sure the solution radioactivity before and
after ion-exchange. 2) Measure the pH dur-
ing a trial labelling: pH after addition of
the first ampoule of NaOH: 8.0 & 0.5. pH
after addition of the ampoule of HCI: 2.0 k
0.5. p H after addition of the second NaOH
ampoule: pH 5 k 1. pH can be measured
with pH paper e.g. Whatman p H paper
(range 1-4 for pH 2.0) and other ranges
with Merck universal paper.
Labelling procedure (time 40 rnin, yield
80-95 per cent)
A kit for albumin labelling consists of:
0.5 ml FeCls/ascorbic acid solution in a
vial, 2 ampoules NaOH, 1 ampoule HCI,
and 1 ion-exchange resin column, as well as
0.10 ml 20 per cent albumin (20 mg) with-
drawn in sterile conditions from a stabilized
20 per cent human serum albumin solution
for infusion (e.g. from Statens Seruniinsti-
tut, Copenhagen).
Add 4 ml 99nlTc eluate (5-20 mCi) to
FeCls/ascorbic acid in a vial (it may be
necessary to dilute the 99"lTc eluate).
While the vial is shaken add one ampoule
NaOH, 0.10 ml 20 per cent albumin and,
slowly, one ampoule HCI. Shake the vial for
15 min (mechanical shaker). Add the second
ampoule NaOH. Remove the whole volume
(5-6 ml) and transfer it to the ion-exchange
resin in the column. A cannula is used as an
air vent. Shake the column for 10 min
(mechanical shaker). 9BnlTc albumin is re-
moved with a short cannula (1 cm) from
the lower end of the ion-exchange column.
A cannula in the top end is used as an air
vent. Control the pH, it should be 5 k 1.
If required sterile filtration (Millipore filter
0.22 pm). The yield may be controlled by
measuring the total activity in the column

(ion-exchange resin plus solution) and the
activity in the total solution removed from
the column.
eemT~-LABELLEDMACROALBUMIN
The preparation of macroalbumin (4, 9, 13, 14)
is very simple in principle. Albumin is precipi-
tated at its isoelectric point (pH approx. 5) by
denaturation with a combination of suitable salt
concentration and heating (80 "C). The size of
the particles of albumin can be controlled by
Downloaded by [RMIT University Library] at 22:09 06 April 2016
using a suitable heating time and shaking, so
that the distribution of particle-size lies in the
range 10-50 pm. The control of precipitation is
critical, because the particles can easily become
too large (several hundred pm).
When an attempt is made to stop the growth
of particles by stopping the procedure pre-
maturely, denaturation is so poor that the par-
ticles resolve as a colloid. If this product is
used for lung scintigraphy, a large part of the
activity is found in the liver.
Certain commercially prepared 1131-labelled
macroalbumins often contain threads up to
several mm. The formation of threads can be
restrained by the addition of a small quantity
of Tween 80 (13). However, this does not solve
the problem of controlling particle-size. The
important factors which influence the forma-
tion of particles and the degree of denaturation
are temperature, heating time, salt and albumin
concentration, and the preliminary treatment of
albumin.
Studies on the labelling procedure
Temperature. Experiments showed that with
a suitable salt concentration albumin could
be precipitated in the temperature range 70-
84 "C reasonably quickly. At 82-84 "C pre-
cipitation was so fast that the size of particles
was large and uncontrollable. At 70-76 "C the
correct particle-size was obtained, but the de-
gree of denaturation was poor, even after ex-
tended heating time. This was demonstrated as
follows. Only the first lung scintigraphy im-
mediately after preparation was satisfactory;
subsequently scintigraphy showed increasing ac-
tivities in the liver. A microscopic comparison
99mTc-Labelling Procediires 3 17
of the distribution of particle-sizes in samples
immediately after preparation and after 1 to 3
hours, showed an increase in small particles in
the latter.
At about 80 "C a stable product with the
required particle-size was occasionally obtained,
but heating often had to be stopped too early
because larger particles (> 50 Itm) were formed,
and denaturation was consequently poor.
Salt concentration. Experiments showed that
both the time taken for particles to be formed
and the size of particles was dependent on salt
concentration (from physiological saline to 0.4
molar sodium acetate in physiological saline).
The degree of denaturation was on the other
hand quite independent of salt concentration.
The immediate conclusion was, therefore, that
in order to obtain a suitable degree of denatura-
tion, the albumin must be heated for at least
10 min at SO "C. With this condition, it was not
possible to find a salt concentration at which
the correct particle-size could be obtained with
every batch.
One solution to this problem was to centri-
fuge the macroalbumin for a very short time,
so that only the largest particles (> 50 blm)
were centrifuged down. The supernatant was
then removed and upon examination had a
better particle-size distribution. However this
method was not suitable for routine use.
The best solution was to divide the denatura-
tion of the albumin into two stages. This takes
a little longer, but the method is considerably
more controllable. The albumin was first pre-
cipitated as particles a little smaller than re-
quired, by a combination of salting out and
heat denaturation (80 "C). Particle-size was
controlled carefully; the heating stage was
terminated (normally after 2-6 min) when the
particles could only just be seen against a light
background. The particle size at this stage was
10-40 im. After cooling the product was cen-
trifuged and the supernatant pipetted off. De-
naturation was then completed by heating
(80 "C) for 10 min in physiological saline. It
was shown that the size of particles did not lin-
crease nearly so quickly in a physiological salt
solution. It was therefore not necessary to con-
trol particle size during this stage of heating,
 318 P . Briricrz
the suspension was simply shaken for 10 rnin
at 80 OC.
If the particles are still too large, their size
can be reduced by a short, energetic shake.
Preparation of reagent (unlimited s!ability)
10 ml Tween 80 (from Fluka) are added to
25 ml sterile, pyrogen-free, physiological saline
and the mixture heated and mixed 2-3 min at
50-60 OC. When the mixture is homogeneous,
it is poured into 975 rnl sterile and pyrogen-
free physiological saline. 1 1 g sodium acetate
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(CHSCOONa, 3 H2O) (p.a.) are added. 10 ml
portions of the solution are dispensed into 20
ml vials. (The size of the vials is chosen so that
they can be put directly into a centrifuge.) The
vials are stoppered (rubber vial diaphragm) and
sterilized.
Labelling procedure (time 25 niin, yield 50-80
per cent)
5-6 ml of galllTcalbumin (5-15 mCi, approx.
20 mg albumin) are added in sterile condi-
tions to 10 ml of sodium acetate reagent in
a vial. The mixture is heated to 80 OC under
mechanical shaking. The formation of albu-
min particles is carefully controlled: at first
the solution is milky, but gradually the
particles become visible when the vial is held
up against a light background. When the par-
ticles can just be seen, the size is 10-40 pm.
Heating has at this point taken 2-6 min. The
vial is cooled to room temperature in a cold
water bath and then centrifuged (5 rnin at 3000
r.p.m.). The supernatant is removed carefully,
using a syringe. A cannula is introduced into
the disphragm as an air vent. 10 ml sterile
physiological saline are added to the albumin.
The product is shaken and heated for 10 min
at 80 "C. Particle-size remains the same or in-
creases slightly. If the particle-size becomes too
large, it is due to ineffective shaking. The vial
is then well shaken by hand after the 10 min
heating. The vial is cooled to rooAmtempera-
ture in a cold water bath.
The distribution of particle-size is controlled
in a Biirker-Tlirk counting chamber under the
microscope. The number of particles per unit
area is counted and placed for example in
> 100, 100, 50, 25, 10, < 10 pcm categories. If
the distribution of particles lies within 10-50
pn, the product is suitable for lung scintig-
raphy. Scintigraphy is carried out immediately
after intravenous injection of 0.6 mCi.
Comments: The time taken for the first pre-
cipitation cannot be determined exactly. For
example, the time varies with differences in the
pretreatment of albumin. If the liver is seen in
lung scintigraphy, the particles are too small
and there is perhaps colloidal albumin in the
product. The rest of the product can be im-
proved by a further 10 rnin heating at 80 OC.
!'!'*LLT~
l R O N ASCORBIC ACID COMPLEX
The preparation of 991nTc iron ascorbic acid
complex (7, 15) is the same as that described
for the preliminary stage of @9111Tc-labelling of
albumin: 9 9 n 1 T ~eluate is added to a solution
of ferric chloride and ascorbic acid. The mix-
ture is adjusted to pH 7-9 by adding NaOH.
The complex is then purified (7) by adsorption
of Tc04- to an ion-exchange resin.
Studies on the labelling procedure
Reagents. Because the method resembles the
9f""Tc albumin procedure, the same amounts
and concentrations of FeC13 and ascorbic acid
sterile in a vial and 1 molar NaOH in an am-
poule (0.38 ml) were used. This presented no
problems.
Determination of TcO4- content in the prod-
uct. An investigation of the yield before ion-
exchange surprisingly never sho'wed a TcO;
peak on separation with Sephadex G-25 (0.8 X
11 cm) (Fig. 3). The 99Ix1Tciron complex came
in the eluate a little after the liquid front, cor-
responding to a compound of high molecular
weight (3-5000 for G-25). The peak was quite
broad (broader than that of 9gmTc albumin)
and exhibited tailing (Fig. 3). Separation of the
99IllTc complex after ion-exchange also ex-
hibited tailing (Fig. 3). The question was wheth-
er this tailing might contain a small TcO4- peak.
An attempt to obtain a better separation with
a longer Sephadex G-25 column (0.8 X 25 cm)
gave the same result and did not provide an
explanation.

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Fig. 3. Separation of E'nlT~iron complex from
nQmTc04-on Sephadex G-25 (0.8 x 11 cm). Elu-
ant: physiological saline.
0 QnrllTciron complex before ion-exchange,
0 onl1lTc iron complex after ion-exchange,
+ 6n"1T~04-.
It was noticed that if separation was per-
formed immediately after preparation (for ex-
ample % rnin after the addition of NaOH) a
well-defined Tc04- peak was obtained, and its
size was increased with increasing amounts of
9Bn1Tc eluate. After more than 10 min after
preparation this Tc04- peak disappeared (Fig.
4). This demonstrates that the formation of a
9gn1Tc iron complex does not take place in-
stantaneously. In a routine preparation there
was usually a certain delay before the determi-
nation was started, so that the TcO4- peak was
not observed.
A f u r t h x experiment confirmed this hypoth-
esis: a certain amount of TcO4- eluate was
added to a 30 rnin old 9en1Tc iron complex
which showed no TcOl- peak (Fig. 5). The
amount added was 11.2 per cent of the total
activity. Elution on Sephadex G-25 immediately
afterwards showed a well-defined TcO4- peak
(hatched area Fig. 5). This peak was calculated
to be 11.5 per cent of the total, which cor-
responded well with the amount added. The re-
maining curves in Fig. 5 show the same mixture
after 30 and 150 min: even added T c 0 was ~ ascorbic acid in vials and 0.38 ml 1 molar
absorbed to the existing complex. Further ex-
"""'Tc-Labelling Procedures 319
103
5
3
1o2
10 ml eluate
Fig. 4. Separation of Y!J1llT~
iron complex on Seph-
adex G-25 (0.8 X 1 1 cm). Eluant: physiological
saline.
a. Complex forined with 6 ml nnlllTceluate:
@ % min after preparation, 0 20 min after
preparation.
b. Complex formed with 1 ml o n l n Teluate:
~
+ complex '/2 min after preparation.
periments with the addition of smaller amounts
of Tc04- showed that the smallest peak that
could be resolved in a separation immediately
after preparation was 2 per cent of the total.
The final conclusion was, therefore, that
with the amounts of reagents used, the TcOl-
ions correspond maximally to 2 per cent of the
activity 10 min after preparation. Ion-exchange
of the prepared 9BmTc complex is therefore
unnecessary-. if the product is used more than
10-15 min after preparation.
The 99111T~ iron complex can be purified by
ion-exchange. A weak anion-exchange resin, for
example Dowex 3, is recommended. The use of
a strong anion-exchange resin used by some
authors (7) reduces the yield considerably, the
comp!ex itself being bound to the resin (Fig. 6).
This binding can be observed - the brown
colour of the complex is weakened by shaking
with strong anion-exchange resins.
Preparation of reagents (stable 2-3 months)
The same reagents are used as those for
9D:l1Tc albumin preparation: 0.50 ml FeCld
NaOH in ampoules.
 320 P . Bririiiz
10
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CP5
I t-+\&
103
5 2
3 Fig. 6. Q@nlTcion complex in the supernatant as a
lo2
10 ml eluate
Fig. 5. Separation of 99n1T~ iron complex on Seph-
adex G-25 (0.8 X 11 cm). Eluant: physiological
saline.
30 rnin after preparation, 0 % min after addi-
tion of 11.2 % @QnlTc04- to the preparation above.
The hatched area - corresponding to QQmTc04--
was determined to approx. 11.5 %. + 30 min after
addition of @@mTc04-.t~ 150 rnin after addition of
O@~TCO~-.
Labelling procedure (titne 15 min, yield > 98
per cent)
5-10 mCi TcO4- eluate (in maximum 10 ml)
are added under sterile conditions to a vial con-
taining FeCl3/ascorbic acid. 1 ampoule NaOH
is then added to the vial while the contents are
shaken. The preparation is completed by al-
lowing the product to stand for 15 min - the
pH (7-9) may be controlled with p H paper.
9gmTc ANTIMONY SULPHIDE COLLOID
Many radioactive colloids can be used for liver
and lymph node scintigraphy (1, 6, 8, 9, 10, 17,
18). Sulphur colloid, microalbumin, and anti-
5 10 2.0
min
function of shaking time and anion-exchange res-
ins. 1 g dry resin shaken with 6 ml @@mTc iron
complex. Symbols are the same as in Fig. 1.
mony sulphide colloid (SbzS3) may be labelled
with 9en1Tc (1, 6, 8, 9, 10, 18). Of these anti-
mony sulphide is probably the easiest to use
and its preparation easiest to control. Remark-
ably 9DmTc antimony sulphide is not a popular
choice of colloid, possibly because of a fear of
toxic and anaphylactic reactions. This product
has been used by us for the last two years for
liver scintigraphy in more than 400 patients;
often repeatedly in the same patient without
any reaction being registered.
A batch of inactive antimony sulphide col-
loid is prepared in a sufficient quantity for 2-3
months' use and dispensed sterile in vials. The
daily labelling procedure is simply to add 9gn1Tc
eluate to the colloid and to heat it for 45 rnin
at 120 "C.Normally antimony sulphide is very
insoluble and is precipitated by addition of
hydrogen sulphide to antimony solution. I n the
presence of a stabilizer such as polyvinylpyrro-
lidon, antimony sulphide can easily be formed
as a colloid. The colloid is a clear orange-
coloured solution. It is very stable - a colloid
more than a year old was used experimentally
 "9t1LTc-Labellirig
Procedures 321

1001- %
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min
I I I 1

15 30 45 60 2 5 10 20
min
Fig. 7. Yield of OOmTc Sb& colloid on heating at
120 "C as a function of heating time. Fig. 9. QglnTcSb& colloid adsorption on anion-
+ 1 ml 9OnlTc eluate exchange resins as a function of shaking time. % g
3 ml QQmTc eluate + 2 rnl Sb2S3 colloid dry resin shaken with !h rnl Tc Sb& + 5 rnl
0 6 ml QPnlTceluate physiological saline.
0 Dowex 1-x2 (50-100 mesh),
@ Amberlite IRA 400 (20-50 mesh),
with satisfactory results. I n order to keep the 0 Dowex 1-x8 (100-200 mesh),
patient dose of antimony sulphide to a mini- + Dowex 2-x8 (20-50 mesh),
mum, the antimony sulphide colloid (1.6 mg (B Dowex 3 (20-50 mesh).
Sb& in 2 ml in a vial) is labelled by adding at
least 10 mCi 9gn1Tceluate, and the patient re-
ceives 1 mCi of the product.

Studies on the labelling procedure

10 ml eluate
Fig. 8. Separation of 9Q"lTc Sb& colloid from
QQ1nTc04-on Sephadex G-25 (0.8 x 11 cm). The
first peak (93 %, 2-4 rnl eluate) is 'J9mTccolloid.
(Eluant: physiological saline).
6 Scand. 1. clin. Lab. Invest.
A determination of the yield as a function of
time showed (Fig. 7) that by heating at 120 "C
for at least 45 min a yield of more than 85 per
cent could be obtained. Furthermore it was
noticed that addition of large volumes of 9 g m T c
eluate reduced the yield. In practice the volume
of eluate per 2 ml antimony sulphide colloid
(1.6 mg Sb&) should be maximally 2-3 ml.
The yield will then be more than 95 per cent.
The flask is heated in a silicone oil bath in a
fume cupboard with closed front. The risk of
an explosion is minimal (200 vials have been
heated in this way without accident). The yield
is determined by dialysis or Sephadex G-25 sep-
aration (0.8 X 11 cm). Fig. 8 shows an ex-
ample of Sephadex separation.
 322 P. Bruuit
Antimony sulphide colloid can be used
directly for scintigraphy, but if the colloid is
required free of the small percentage of un-
bound 9Bn1Tc, a weak anion-exchange resin
such as Dowex 3 should be used (see Fig. 9).
Dowex 1 adsorbs too much of the colloid. T h e
preparation of the ion-exchange columns is
similar to that for 9B1l1Tc albumin labelling:
2.5 ml portions of Dowex 3 are sterilized in
glass tubes with glass filter disc and rubber
diaphragms in both ends.
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Preparation of reagent (stable 3 months)
Sterilized apparatus and sterile, pyrogen-free
distilled water are used (notice that physiological
saliine 'solution cannot be used). 100 d distilled
water in a 200 ml flask are heated to boiling
point in a water bath. 0.60 g PVP (K 25)
(purum pharm. from Fluka) is dissolved in 10
ml distilled water and added to the 100 ml
boiling water. 0.20 g potassium antimony tar-
trate (K(SbO)C4Ha06,'/2 HeO) is dissolved in 20
ml distilled water and added directly to the
flask using a glass rod. Hydrogen sulphide is
fed into the flask. The temperature is kept a t
approx. 100 "C. T h e hydrogen sulphide feed is
retained (10-1 5 min) until the solution has been
dark orange for a while. T h e solution must be
clear. T h e flask is then heated on a hot plate
with magnetic stirrer and the excess hydrogen
sulphide boiled off. Magnetic stirring is recom-
mended at this stage to avoid the solution's
boiling over. Boiling is continued at least 15
min. Hydrogen sulphide content is controlled
with damp lead acetate paper. A colour-change
to faint brown is allowed. After cooling, the
clear dark orange liquid is dispensed in 2 ml
portions into 20 ml vials and sterilized.
Labelling procedure (time 45 rnin, yield > 95
per cent)
10 mCi 9gmTc eluate (maximum volume
2-3 ml) is added to 2 ml antimony sulphide col-
loid in a vial. T h e vial is heated for 45 m i n at
120 OC in a silicone oil bath (it is recommended
that heating takes place in a fume cupboard
with closed front). After heating the product is
ready.
ACKNOWLEDGEMENT
I wish to thank Mrs Bente Eriksen for valuable
technical assistance.
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Received 15 December 1970
Accepted 28 July 1971
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~q9t’1Tc-Labellirig
Procedures 323
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