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Gelatine-alginate complex gel: A new acoustically tissue-equivalent material
Ultrasoundin Med. & Biol. Vol. 9, No. 5, pp. 479 484, 1983 Printed in Great Britain.
0301 5629/83 $3.00+ .00 Pergamon PressLtd.
GELATINE-ALGINATE COMPLEX GEL: A NEW ACOUSTICALLY TISSUE-EQUIVALENT MATERIAL N. L. BUSH and C. R. HILL Physics Division, Institute of Cancer Research, Royal Marsden Hospital, Sutton, Surrey, England (Received 19 October 1982; in f i n a l f o r m 15 April 1983)
Abstract--Methods are described for the preparation of gelatin~alginate complex gels and measurements are reported of certain of their acoustic and physical properties relevant to their use as tissue-equivalent phantoms in medical ultrasonics applications. Speed o f sound at 20°C is 1520 ms I with a coefficient of + 2.6 ms-I/°C. Attenuation coefficient in unloaded gel is 0.12dBcm t MHz -~ (varying approximately linearly with frequency) but can readily be increased to at least 0.5 dB cm i MHz i by loading the gel with polyethylene or lipid microspheres. Volume stability under conditions of water immersion without an impermeable boundary layer is within about ___2 ~ over a 300 day period, an improvement by a factor of at least 25 on both simple and cross-linked gelatine gels, and stability against chemical or bacterial degradation can also readily be maintained. Key Words: Ultrasound, Phantoms, Gels, Tissue-equivalence.
thermal stability can be achieved by adding cross linking agents such as chrome alum (Sommer et al., 1980) or formaldehyde (Astrahan, 1979) to the gels. Nevertheless, the problem of water uptake or loss has not been satisfactorily resolved. Methods described in the literature (Somer et al., 1980) for controlling water diffusion, employ one or more barrier layers acoustically matched to the gel. In our experience however protective barrier layers are usually not totally effective and, in some applications, can prove to introduce unacceptable perturbations in experimental conditions. Another approach to solution of this problem would be to use a more stable hydrocolloid, namely, microbiological culture medium agar gel (Burlew et al., 1980). However, our experience of this material, and also that of its original proponents (Madsen et al., 1982a), is that it is very difficult to prepare in suitably homogeneous form. We have therefore explored the possibility of stabilizing gels by chemical modification and have proceeded to document the acoustic behaviour of the resulting materials.
INTRODUCTION
There is a recognized need for synthetic materials that can be manufactured in a controlled manner to be approximately equivalent acoustically to particular human and animal soft tissues. In our own work the need to develop improved phantoms has arisen in three contexts: studies of the transfer characteristics of imaging systems, developments of methods of quantitative tissue characterization and studies of the ultrasonic induction of hyperthermia. Outline specifications for such materials will generally include features such as those listed in Table 1. Published work (Edmonds et al., 1979; Eggleton and Whitcomb, 1979) indicates that the choice of materials that will meet the above criteria is very limited, and that those materials which are found to have generally suitable characteristics are nearly all hydrocoloids. Several authors have reported the use of gelatine gels. Madsen et al. (1978), for example, have shown that sound speed in such gels can be controlled by doping with an alcohol, and attenuation coefficient varied by loading with fine graphite powder. However, unmodified gelatine, which is an organic hydrophilic material obtained as a breakdown product of collagen, has the disadvantage of being inherently unstable: It is thermally unstable above its gelling point of 25°C, is susceptible to bacterial and fungal attack and is very sensitive to diffusion of water across the gel surface, producing drastic changes in phantom dimension and acoustic properties. The problems of thermal and biological stability are relatively easy to control, bacterial and fungal attack can be prevented by doping gels with suitable bactericides and fungicides (Madsen et al., 1978), and
METHODS: PREPARATION OF GELATINE-ALGINATE COMPLEXES
Following initial studies with unmodified gelatine, in which water uptake or loss was identified as a major factor leading to instability in acoustic properties, we investigated the properties of another hydrocolloid, algin: a seaweed colloid extracted from brown algae (Kelp: order Laminariales). Algin is a polysaccharide with ionic properties, forming soluble and insoluble salts. The soluble alginates, notably sodium 479
480
N.L. BUSHand C. R. HILL Table. l Exemplaryspecificationslbr tissue equivalent get phantoms Exemplary
specification
(a)
Unloaded
(non-scatterin 9) b a s e
i)
Attenuation
2)
Speed of sound
3)
Internal s c a t t e r i n g reflection
4)
Density
coefficient
gel
O.2 + O.i db cm -I at 1 MHz approx, linear a t t e n u a t i o n frequency dependence,
and
negligible. 1.O5 + O.i q cm -3
5)
Phase Thermal
7)
Temporal against
8)
Ease and e c o n o m y
9)
Repeatability
solid or semi-solid. stability
at up to 65°C.
stability
Loaded
as above
:
gel p h a n t o m s
1540 + 20 ms -I at 37°C (controllable w i t h i n range 1 5 0 0 - 16OO ms-l),
6)
(b)
for t i s s u e - e q u i v a l e n t
b a c t e r i a l / f u n g a l action chemical action (including s c a n n i n g gels) wet and dry storage.
of manufacture.
of properties.
(scattering)
except
soft-tissue-equivalent
for points (i) and
i)
Attenuation
coefficient
3)
Internal s c a t t e r i n g reflection
and
alginate, produce typically viscous colloids with only 2~o wt./vol solution in water. Alginate gels have also been described (McDowell, 1966) but their manufacture is a complex process which involves the slow release of calcium ions into a sodium alginate solution. The resulting gels are a mixture of insoluble calcium alginate and sodium alginate, with the proportion of insoluble to soluble salt determining the gel strength (i.e. rigidity increases proportionally with [Ca]). For our purposes an important property of sodium alginate is its ability to complex with gelatine and thus form a more stable system (Le Gloahec, 1947). The formation of a molecularly more complex compound was reported by Le Gloahec (1947) to occur when sodium alginate and gelatine solutions were mixed under specific conditions of pH and concentration. Our study investigated the physical properties of this material, in a gelled state, to assess any advantages of this complex gel over gelatine gels, particu-
9el
(3) as follows:
:
0.7 + 0.3 db cm -I at 1 MHz approx, linear a t t e n u a t i o n frequency depencence.
:
simulating scattering b e h a v i o u r of n o r m a l and p a t h o l o g i c a l soft tissues.
larly for water uptake. The gelatine alginate complex was reproduced in varying ratios of 16~owt./vol gelatine and 2~o wt./vol sodium alginate solutions at 35~'C and pH 3-5 (pH specified as between gelatine and alginate isoelectric points (Le Gloahec, 1947)). The complex solutions produced cloudy colloidal suspensions that, when cooled below 25°C, formed opaque gels. The principle interest in these gels was to see if the complexing reaction reduced the number of reactive hydroxide residues for water uptake, and to determine whether, like alginate solutions, the gels were ionic. If so, a further reduction in solubility would be achieved by combining the gel with an insoluble alginate ion such as calcium. A preliminary evaluation of the water uptake and temperature stability of the gel complex was provided by experimentally storing, under water, the phantoms of varying component ratios. The phantoms were stored at temperatures between 4 and 60°C and half were pretreated in CaCI2 to check the efficacy of Ca ion
Gelatine-alginate complex gel
treatment. Notably when the complex gels are compared with pure gelatine, water uptake is lower, and CaCI2 treatment also improves thermal stability, providing irreversible gellation. The best gel ratio for manufacture and storage was found to be a 50/50 mix of the component solutions. By varying the pH of the gelatine alginate reaction three distinct phases were distinguished. Between pH 3 and 5 the mixture produced is cloudy and variable but gels when cooled; this is caused by the complex coagulate being held in colloidal suspension by unreacted gelatine or alginate. At pH less than 3 the reaction produces a clear supernatant and a coagulate (the coagulate-algino gelatine, if dried and resuspended, would not gel). Finally at pH values above 5-7 a clear homogeneous aggregate that gels when cooled is formed. This gel is probably not the product of a condensation reaction; however, it does have the advantage of clarity, repeatability and low water uptake (after CaC12 treatment). So from our preliminary studies the best base gel formula was the following: 16~owt./vol gelatine solution, 2 ~ wt./vol sodium alginate solution combined in a 1:1 ratio at 35°C, and buffered at pH 6-7, plus 0.2~o Tolvic acid as an antibacterial preservative. The solution was gelled by cooling the mixture below 25°C and finally rendered thermally stable, irreversibly, by CaCI 2 treatment (0.5 M, 24 hr). This gel, while displaying similar physical properties to gelatine gels, showed much increased thermal stability as compared with non-crosslinked gelatine and minimal water uptake when stored under water at normal experimental temperatures (4-30°C). Using methods described by Bamber et al. (1977) we measured the speed of sound in the gels and their attenuation properties over a range of temperatures. A measure of the repeatability of the gel properties was obtained by monitoring the properties of control gel samples for each new gel batch made. All measurements were made on barrier-less gel samples moulded into cylinders of identical shape and size (6.5cmx 4.5cmdia), measurements being made along the axis of the cylinder, between the flat parallel-faced ends. The first phantoms produced were simple constructions designed for basic studies on interactions between sound and tissues. In these models the base gel was to act as a matrix into which scattering particles of varying size, strength and number could be randomly but evenly distributed. Scattering particles which have been used are: Sephadex, polyethylene and polystyrene beads, plus suspensions of bovine fat particles. The choice of particles was determined by two factors: impedance mismatch with the base
481
gel and, secondly, particle size (when considering sizes the standard deviation of the distribution is important). The main series of measurements was made using polyethylene beads of diameter 120 #m +__28/~m; Z = 1.866 x 106 Rayl; p = 0.93 g/ml, which gave medium strength echoes. Phantoms made using these scatterers used the number of scatterers per unit volume as their principle variable; their use is explained in greater detail by Hall et al. (1982) and Merton et al. (1982). Sephadex beads, in contrast to polyethylene beads, show a very small impedance mismatch in gel and accordingly their echoes are very weak. Two additional series of measurements were made using respectively polyethylene spheres less homogeneous in size distribution (119/~m unweighted mean diameter measured on a Vernier microscope graticule, ___58/~m S.D.) and bovine peritoneal fat particles (40 #m + 25 ~tm). To manufacture these simple monoscatter phantoms the major problems to avoid are the introduction of air bubbles and scatterer gradients. To achieve this the pure gel is first degassed, then the hot gel mixture is added to the scatterers in a sealed container (mould). The number of scatterers was calculated by weight, given the known densi.ty and mean volume of the particles. The gel and scatterers are mixed randomly by agitating the container, whilst ensuring that all air bubbles are excluded. Finally the phantom is cooled at 4°C while slowly rotating the cylindrical phantom with its axis horizontal (Madsen et al., 1978), to prevent any settling of particles during gellation (the direction and speed of any settling depends on the density mismatch between scatterers and gel). Once properly gelled the phantom is removed from its mould and suspended for 24 hr in CaC12 solution in order to ensure increased stability. All phantoms produced were stored under water at 4°C, in closed containers. To prevent decay a small quantity of Roccal (a water bath disinfectant; Winthrop Laboratories, Surbiton, Surrey, U.K.) was added to the water. A required characteristic of the base gel is its ability to produce composite phantoms (i.e. phantoms with inclusions of different scattering strength, type or size). Complex phantom designs are made possible by the irreversible nature of the gel once treated in CaC12, so pretreated gel inclusions will remain in the gel phase when potted in hot gel. This is, for some purposes, a significant improvement on the procedure described and employed previously (Madsen et al., 1980, 1982b, 1982c, 1982d) which simply exploits a difference between the melting and congealing temperatures of the gel. An example of such complex
482
N.L. BUSHand C. R. HILL
phantoms that we have constructed is given by a constant resolution phantom produced with four conical inserts of identical size but different polyethylene scatterer concentrations; 4/mm 3, 6/mm 3, 8/mm 3, 12/mm 3 embedded in an outer matrix of scatterer concentrations 4/mm 3 (Fig. 4). Another phantom construction employing our base gel was used for investigating heating profiles obtained when irradiating a muscle equivalent gel (acoustic absorption values as for muscle) with therapeutic intensities of ultrasound (ter Haar and Carnochan, 1981). The actual construction used was a pure gelatine alginate gel block into which an array of thermocouples was prepotted. RESULTS: ACOUSTIC AND PHYSICAL PROPERTIES OF GELATINE-ALGINATE COMPLEXES
Records of the observation of dimensional (volume) stability of both the simple and complexed gels, under various wet and dry storage conditions, are shown in Fig. I. These clearly show the radical improvement of stability in this respect consequent on the complexing process. Measured sound speed in the gelatine-alginate complex (90.75% waterw/w) is 1519+ 1.2ms i at 20~C and variation with temperature in the range 1 7 . Y ' C - 2 5 ° C is linear with a coefficient of 2.6 ms-Z/~C. From a limited number of observations on the gel complex loaded with 120/~m polyethylene spheres up to concentration of 10 scatterers mm 3
there appears to be little dependence of sound speed on such loading. Corresponding results for graphite powder in gelatine gels have been reported by Madsen (1980) and in agar gels by Burlew et al. (1980). Figures 2 and 3 indicate the measured variations of attenuation coefficient with frequency observed both for the pure gel complex and for gels loaded with either 120 # m polyethylene spheres (Fig. 2) or 40/~m dia. particles of bovine peritoneal fat (Fig. 3). The corresponding data for a pure gelatine gel (90% water w/w) are also shown in Fig. 2 for comparison. DISCUSSION
The gelatine alginate gel provides a readily available and usable base material with properties that, combined with the introduction of scattering particles, allow for a whole variety of simple through to complex phantom constructions. The gel's ability to be stored under water, without need for elaborate protection, showing only minimal water uptake for periods of 6 months or longer, whilst maintaining its initial properties, is a considerable advantage over both simple and cross-linked gelatine gels. It is important, however, that the desirable properties of gelatine be maintained, and particularly the ease with which it enables manufacture of uniform, reproducible acoustic properties. So it is encouraging to note that a 10% gelatine gel, with comparable water levels to our new gel, also has very similar acoustic properties to the gel alginate material.
6O 2o
~,zo Storage Conditions:
'/"/~>'~ 200
/-,c
o Dry ) Gelatine z~ WaterJ 16% [] Water, Gelatine 16% + 0.5% Formalin o Water~ Gel/Alginate
2C c(D
1ot 2o/ \
~30~
N
Storage Time (days)
20o
300
Fig. 1. Measured weight change in 60 ml gelatine 16% wt./vol gel and gelatine-alginategel phantoms vs time stored under various conditions. Note that under "dry" conditions the weight-loss curve for gelatine-alginate complexis similar to that shown for pure gelatine./k, Gelatine immersedin water, 2ff~C. A, Gelatine immersedin water, 4°C. El, Gelatine plus 0.5% formaldehyde,immersed in water, 20°C. ~, Gelatine dry, 20"C. 0, Gelatine dry, 4°C. Gelatine-alginate immersed in water, 4°C. ©, Batch 1. Q, Batch 2.
Gelatine-alginate complex gel
483
Oet/Algir~te tooded with Polyethylene scatterers: [[) graded 120/Jm ~[)ungraded 119/Jm
10 g 6
/ oE
//"
10%
~ (I) o/12,5mm-3
/
Human Liver
/
¢-
/
._o ~5
/I
¢-
/"
,~ +
/Z'
1,
~ 4 ,,r)>75mm,,m _-,
•J /
Q/Q " g
/,/
//i,,
/ 1//ol
,.D-- .~'~i ~ ~ "
2 ~
o_~o~o~°~°~
~. . . . . . . . . . . . . .
~-
3
.---'/
GIA
oetatlne ~o%
5 6 i 8 § lb Frequency(MHz) Fig. 2. Measured ultrasonic attenuation coefficient vs. frequency for pure and polyethylene loaded gelatine-alginate gels, at 22°C. E], Pure gelatine, 10%wt./vol, 90% waterw/w, O, pure gelatine-alginate. +, human liver (Bamber and Hill, 1979). Gelatine-alginate loaded with graded polyethylene 120/~m scatterers. i , 1666 scatterers cm -3. V, 3750 scatterers cm- 3. i,, 10000 scattererscm 3. O, 12500 scattererscm -3. Gelatine-alginate loaded with ungraded polyethylene l l9~um scatterers. A, 1666 scatterers cm 3. ~ , 3750 scatterers cm- 3. O
/
/ i
///Jiii:o
A
W h e n studying the acoustic properties o f the new gel, it is i m p o r t a n t to note the variation o f properties as a function o f temperature. We measured this variation for velocity against temperature and, at 22°C, dc/dt = 2 . 6 m s - ~ / ° C : this m a y be c o m p a r e d with a value o f 1.8 m s - ~/°C for h u m a n liver tissue at 20°C as measured by Bamber and Hill (1979). D a t a for attenuation coefficient as a function o f scatterer concentrations and size, measured in gel p h a n t o m s using two different scatterer types (Figs. 2 and 3), as expected d e m o n s t r a t e d increased attenutation with higher scatterer concentrations. One interesting feature observed in these results was the effect o f scatterer size. The graded 120/am polyethylene particles produced a frequency related effect (evident as an inflection in the attenuation/frequency curve, irrespective o f concentration) at 4 M H z ; this feature was absent however in curves obtained either with the more heterogeneous polyethylene particles or with the bovine fat particles, which suggests that a frequency effect related to scatterer size is being d e m o n s t r a t e d with the graded particles. Clearly such an effect m a y need to be avoided for some applications and, with
Frequency(HHz) Fig. 3. Measured ultrasonic attenuation coefficient vs. frequency for gelatine-alginate gels loaded with various concentrations of bovine peritoneal fat (E particle dia. = 40/~m), at 22°C. O, Pure gelatine-alginate. Gelatine-atginate loaded with bovine peritoneal fat globules. O, 0.5% w/w. ~, 1%w/w./k, 2.5%w/w. [--], 5% w/w. O, 10% w/w.
A
B
C
D
*-3cm-4' Fig. 4. A B-scan image of a polyethylene loaded gel-alginate phantom. The phantom comprises four conical inserts embedded in a surrounding matrix, the polyethylene scatterer concentration in the matrix was 4/mm3and the inserts. (A) 4/mm 3. (B) 6/mm3. (C) 12/mm3. (D) 16/mm3. this proviso, the results presented here indicate that p h a n t o m s o f the type described can be m a d e to provide a g o o d m a t c h to h u m a n liver in terms o f frequency dependent attenuation coefficient.
Acknowledgements--Weare grateful to J. C. Bamber for help and advice with the attenuation measurements and to other colleagues for helpful discussions. The work was carried out with funds provided jointly by Medical Research Council and the Cancer Research Campaign.
484
N . L . BUSH and C. R. HILL REFERENCES
Astrahan M. A. (1979) Concerning hyperthermia phantom.Med. Phys. 6, 235. Bamber J. C., Fry M. J., Hill C. R. and Dunn F. (1977) Ultrasonic attenuation and backscattering by mammalian organs as a function of time after excision. Ultrasound in Med. & Biol. 3, 15-20. Bamber J. C. and Hill C. R. (1981) Acoustic properties of normal and cancerous human liver-l. Dependence on pathological condition. Ultrasound in Med. & Biol. 7, 121-133. Bamber J. C. and Hill C. R. (1979) Ultrasonic attenuation and propagation speed in mammalian tissues as a function of temperature. Ultrasound in Med. & Biol. 5, 149-157. Burlew M. M., Madsen E. L., Zagzebski J. A., Banjavic R. A. and Sum S. (1980) A new ultrasound tissue equivalent material. Radiology 134, 517-520. Edmonds P. D., Reyes Z., Filly R. A., Parkinson D. B. and Busey H. (1979) A human abdominal tissue phantom. In Ultrasonic tissue characterisation H(Edited by M. Linzer), pp. 323-326. NBS Spec. Publ. 525, Washington D. C. Eggleton R. C. and Whitcomb J. A. (1979) Tissue simulators for diagnostic ultrasound. In Ultrasonic Tissue Characterisation II (Edited by M. Linzer), pp. 327-336. NBS Spec. Publ. 525, U.S. Govt. Printing Office. Hall T. J., Bamber J. C. and Hill C. R. (1982) The influence of transducer characteristics and signal processing on fine structure in ultrasound images. Ultrasound in Med. Biol. 8 (Suppl. 1), 72. Le Gloahec V. C. R. (1947) Proteinous compound and the manufacture thereof. U.S. Pat. No. 2430, 180, 4 November. Madsen E. L., Zagzebski J. A., Banjavic R. A. and Jutila R. E. (1978) Tissue mimicking materials for ultrasound phantoms. Med. Phys. 5, 391-394. Madsen E. L. (1980) Ultrasonically soft-tissue mimicking materials.
In Medical Physics of CT and Ultrasound: Tissue imaging and characterisation (Edited by G. D. Fullerton and J. A. Zagzebski), pp. 531-550. American Institute of Physics, New York. Madsen E. L., Zagzebski J. A. and Frank G. R. (1982A) Oil in gelatin dispersions for use as ultrasonically tissue-mimicking materials. Ultrasound in Med. & Biol. 8, 277-287. Madsen E. L., Zagzebski J. A., Frank G. R., Greenleaf J. F. and Carson P. L. (1982b) Anthropomorphic breast phantoms for assessing ultrasonic imaging system performance and for training ultrasonographers--l. J. Clin. Ultrasound 10, 67-75.--II. J. Clin. Ultrasound 10, 91-100. Madsen E. L., Zagzebski J. A. and Ghilardi-Netto T. (1980) An anthropomorphic torso section phantom for ultrasonic imaging. Med. Phys. 7, 43-50. Madsen E. L., Zagzebski J. A. and Frank G. R. (1982d) An anthropomorphic ultrasound breast phantom containing intermediate-sized scatterers. Ultrasound in Med. & Biol. 8, 381 392. McDowell R. H. (1966) Alginates and their derivatives. Society of Chemical Industry, Monograph No. 24. The Chemistry and Rheology of water soluble gums and Colloids, pp. 19-32, SCI, 14 Belgrave Sq., London SWI. Merton J., Nicholas D. and Hill C. R. (1982) An assessment of the use of frequency and orientation diffraction for identifying scatterer concentration in models. Ultrasound in Med. & Biol. 8 (Suppl. 1), 128. Sommer F. G., Filly R. A., Edmonds P. D., Reyes Z. and Comas H. E. (1980) A phantom for imaging biological fluids by ultrasound and CT scanning. Ultrasound in Med. & Biol. 6, 135-140. tcr Haar G. and Carnochan P. (1982) A comparison of ultrasonic irradiation and RF inductive heating for clinical localised hyperthermia applications. Br. J. Cancer 45, Suppl. V, 77.
0301 5629/83 $3.00+ .00 Pergamon PressLtd.
GELATINE-ALGINATE COMPLEX GEL: A NEW ACOUSTICALLY TISSUE-EQUIVALENT MATERIAL N. L. BUSH and C. R. HILL Physics Division, Institute of Cancer Research, Royal Marsden Hospital, Sutton, Surrey, England (Received 19 October 1982; in f i n a l f o r m 15 April 1983)
Abstract--Methods are described for the preparation of gelatin~alginate complex gels and measurements are reported of certain of their acoustic and physical properties relevant to their use as tissue-equivalent phantoms in medical ultrasonics applications. Speed o f sound at 20°C is 1520 ms I with a coefficient of + 2.6 ms-I/°C. Attenuation coefficient in unloaded gel is 0.12dBcm t MHz -~ (varying approximately linearly with frequency) but can readily be increased to at least 0.5 dB cm i MHz i by loading the gel with polyethylene or lipid microspheres. Volume stability under conditions of water immersion without an impermeable boundary layer is within about ___2 ~ over a 300 day period, an improvement by a factor of at least 25 on both simple and cross-linked gelatine gels, and stability against chemical or bacterial degradation can also readily be maintained. Key Words: Ultrasound, Phantoms, Gels, Tissue-equivalence.
thermal stability can be achieved by adding cross linking agents such as chrome alum (Sommer et al., 1980) or formaldehyde (Astrahan, 1979) to the gels. Nevertheless, the problem of water uptake or loss has not been satisfactorily resolved. Methods described in the literature (Somer et al., 1980) for controlling water diffusion, employ one or more barrier layers acoustically matched to the gel. In our experience however protective barrier layers are usually not totally effective and, in some applications, can prove to introduce unacceptable perturbations in experimental conditions. Another approach to solution of this problem would be to use a more stable hydrocolloid, namely, microbiological culture medium agar gel (Burlew et al., 1980). However, our experience of this material, and also that of its original proponents (Madsen et al., 1982a), is that it is very difficult to prepare in suitably homogeneous form. We have therefore explored the possibility of stabilizing gels by chemical modification and have proceeded to document the acoustic behaviour of the resulting materials.
INTRODUCTION
There is a recognized need for synthetic materials that can be manufactured in a controlled manner to be approximately equivalent acoustically to particular human and animal soft tissues. In our own work the need to develop improved phantoms has arisen in three contexts: studies of the transfer characteristics of imaging systems, developments of methods of quantitative tissue characterization and studies of the ultrasonic induction of hyperthermia. Outline specifications for such materials will generally include features such as those listed in Table 1. Published work (Edmonds et al., 1979; Eggleton and Whitcomb, 1979) indicates that the choice of materials that will meet the above criteria is very limited, and that those materials which are found to have generally suitable characteristics are nearly all hydrocoloids. Several authors have reported the use of gelatine gels. Madsen et al. (1978), for example, have shown that sound speed in such gels can be controlled by doping with an alcohol, and attenuation coefficient varied by loading with fine graphite powder. However, unmodified gelatine, which is an organic hydrophilic material obtained as a breakdown product of collagen, has the disadvantage of being inherently unstable: It is thermally unstable above its gelling point of 25°C, is susceptible to bacterial and fungal attack and is very sensitive to diffusion of water across the gel surface, producing drastic changes in phantom dimension and acoustic properties. The problems of thermal and biological stability are relatively easy to control, bacterial and fungal attack can be prevented by doping gels with suitable bactericides and fungicides (Madsen et al., 1978), and
METHODS: PREPARATION OF GELATINE-ALGINATE COMPLEXES
Following initial studies with unmodified gelatine, in which water uptake or loss was identified as a major factor leading to instability in acoustic properties, we investigated the properties of another hydrocolloid, algin: a seaweed colloid extracted from brown algae (Kelp: order Laminariales). Algin is a polysaccharide with ionic properties, forming soluble and insoluble salts. The soluble alginates, notably sodium 479
480
N.L. BUSHand C. R. HILL Table. l Exemplaryspecificationslbr tissue equivalent get phantoms Exemplary
specification
(a)
Unloaded
(non-scatterin 9) b a s e
i)
Attenuation
2)
Speed of sound
3)
Internal s c a t t e r i n g reflection
4)
Density
coefficient
gel
O.2 + O.i db cm -I at 1 MHz approx, linear a t t e n u a t i o n frequency dependence,
and
negligible. 1.O5 + O.i q cm -3
5)
Phase Thermal
7)
Temporal against
8)
Ease and e c o n o m y
9)
Repeatability
solid or semi-solid. stability
at up to 65°C.
stability
Loaded
as above
:
gel p h a n t o m s
1540 + 20 ms -I at 37°C (controllable w i t h i n range 1 5 0 0 - 16OO ms-l),
6)
(b)
for t i s s u e - e q u i v a l e n t
b a c t e r i a l / f u n g a l action chemical action (including s c a n n i n g gels) wet and dry storage.
of manufacture.
of properties.
(scattering)
except
soft-tissue-equivalent
for points (i) and
i)
Attenuation
coefficient
3)
Internal s c a t t e r i n g reflection
and
alginate, produce typically viscous colloids with only 2~o wt./vol solution in water. Alginate gels have also been described (McDowell, 1966) but their manufacture is a complex process which involves the slow release of calcium ions into a sodium alginate solution. The resulting gels are a mixture of insoluble calcium alginate and sodium alginate, with the proportion of insoluble to soluble salt determining the gel strength (i.e. rigidity increases proportionally with [Ca]). For our purposes an important property of sodium alginate is its ability to complex with gelatine and thus form a more stable system (Le Gloahec, 1947). The formation of a molecularly more complex compound was reported by Le Gloahec (1947) to occur when sodium alginate and gelatine solutions were mixed under specific conditions of pH and concentration. Our study investigated the physical properties of this material, in a gelled state, to assess any advantages of this complex gel over gelatine gels, particu-
9el
(3) as follows:
:
0.7 + 0.3 db cm -I at 1 MHz approx, linear a t t e n u a t i o n frequency depencence.
:
simulating scattering b e h a v i o u r of n o r m a l and p a t h o l o g i c a l soft tissues.
larly for water uptake. The gelatine alginate complex was reproduced in varying ratios of 16~owt./vol gelatine and 2~o wt./vol sodium alginate solutions at 35~'C and pH 3-5 (pH specified as between gelatine and alginate isoelectric points (Le Gloahec, 1947)). The complex solutions produced cloudy colloidal suspensions that, when cooled below 25°C, formed opaque gels. The principle interest in these gels was to see if the complexing reaction reduced the number of reactive hydroxide residues for water uptake, and to determine whether, like alginate solutions, the gels were ionic. If so, a further reduction in solubility would be achieved by combining the gel with an insoluble alginate ion such as calcium. A preliminary evaluation of the water uptake and temperature stability of the gel complex was provided by experimentally storing, under water, the phantoms of varying component ratios. The phantoms were stored at temperatures between 4 and 60°C and half were pretreated in CaCI2 to check the efficacy of Ca ion
Gelatine-alginate complex gel
treatment. Notably when the complex gels are compared with pure gelatine, water uptake is lower, and CaCI2 treatment also improves thermal stability, providing irreversible gellation. The best gel ratio for manufacture and storage was found to be a 50/50 mix of the component solutions. By varying the pH of the gelatine alginate reaction three distinct phases were distinguished. Between pH 3 and 5 the mixture produced is cloudy and variable but gels when cooled; this is caused by the complex coagulate being held in colloidal suspension by unreacted gelatine or alginate. At pH less than 3 the reaction produces a clear supernatant and a coagulate (the coagulate-algino gelatine, if dried and resuspended, would not gel). Finally at pH values above 5-7 a clear homogeneous aggregate that gels when cooled is formed. This gel is probably not the product of a condensation reaction; however, it does have the advantage of clarity, repeatability and low water uptake (after CaC12 treatment). So from our preliminary studies the best base gel formula was the following: 16~owt./vol gelatine solution, 2 ~ wt./vol sodium alginate solution combined in a 1:1 ratio at 35°C, and buffered at pH 6-7, plus 0.2~o Tolvic acid as an antibacterial preservative. The solution was gelled by cooling the mixture below 25°C and finally rendered thermally stable, irreversibly, by CaCI 2 treatment (0.5 M, 24 hr). This gel, while displaying similar physical properties to gelatine gels, showed much increased thermal stability as compared with non-crosslinked gelatine and minimal water uptake when stored under water at normal experimental temperatures (4-30°C). Using methods described by Bamber et al. (1977) we measured the speed of sound in the gels and their attenuation properties over a range of temperatures. A measure of the repeatability of the gel properties was obtained by monitoring the properties of control gel samples for each new gel batch made. All measurements were made on barrier-less gel samples moulded into cylinders of identical shape and size (6.5cmx 4.5cmdia), measurements being made along the axis of the cylinder, between the flat parallel-faced ends. The first phantoms produced were simple constructions designed for basic studies on interactions between sound and tissues. In these models the base gel was to act as a matrix into which scattering particles of varying size, strength and number could be randomly but evenly distributed. Scattering particles which have been used are: Sephadex, polyethylene and polystyrene beads, plus suspensions of bovine fat particles. The choice of particles was determined by two factors: impedance mismatch with the base
481
gel and, secondly, particle size (when considering sizes the standard deviation of the distribution is important). The main series of measurements was made using polyethylene beads of diameter 120 #m +__28/~m; Z = 1.866 x 106 Rayl; p = 0.93 g/ml, which gave medium strength echoes. Phantoms made using these scatterers used the number of scatterers per unit volume as their principle variable; their use is explained in greater detail by Hall et al. (1982) and Merton et al. (1982). Sephadex beads, in contrast to polyethylene beads, show a very small impedance mismatch in gel and accordingly their echoes are very weak. Two additional series of measurements were made using respectively polyethylene spheres less homogeneous in size distribution (119/~m unweighted mean diameter measured on a Vernier microscope graticule, ___58/~m S.D.) and bovine peritoneal fat particles (40 #m + 25 ~tm). To manufacture these simple monoscatter phantoms the major problems to avoid are the introduction of air bubbles and scatterer gradients. To achieve this the pure gel is first degassed, then the hot gel mixture is added to the scatterers in a sealed container (mould). The number of scatterers was calculated by weight, given the known densi.ty and mean volume of the particles. The gel and scatterers are mixed randomly by agitating the container, whilst ensuring that all air bubbles are excluded. Finally the phantom is cooled at 4°C while slowly rotating the cylindrical phantom with its axis horizontal (Madsen et al., 1978), to prevent any settling of particles during gellation (the direction and speed of any settling depends on the density mismatch between scatterers and gel). Once properly gelled the phantom is removed from its mould and suspended for 24 hr in CaC12 solution in order to ensure increased stability. All phantoms produced were stored under water at 4°C, in closed containers. To prevent decay a small quantity of Roccal (a water bath disinfectant; Winthrop Laboratories, Surbiton, Surrey, U.K.) was added to the water. A required characteristic of the base gel is its ability to produce composite phantoms (i.e. phantoms with inclusions of different scattering strength, type or size). Complex phantom designs are made possible by the irreversible nature of the gel once treated in CaC12, so pretreated gel inclusions will remain in the gel phase when potted in hot gel. This is, for some purposes, a significant improvement on the procedure described and employed previously (Madsen et al., 1980, 1982b, 1982c, 1982d) which simply exploits a difference between the melting and congealing temperatures of the gel. An example of such complex
482
N.L. BUSHand C. R. HILL
phantoms that we have constructed is given by a constant resolution phantom produced with four conical inserts of identical size but different polyethylene scatterer concentrations; 4/mm 3, 6/mm 3, 8/mm 3, 12/mm 3 embedded in an outer matrix of scatterer concentrations 4/mm 3 (Fig. 4). Another phantom construction employing our base gel was used for investigating heating profiles obtained when irradiating a muscle equivalent gel (acoustic absorption values as for muscle) with therapeutic intensities of ultrasound (ter Haar and Carnochan, 1981). The actual construction used was a pure gelatine alginate gel block into which an array of thermocouples was prepotted. RESULTS: ACOUSTIC AND PHYSICAL PROPERTIES OF GELATINE-ALGINATE COMPLEXES
Records of the observation of dimensional (volume) stability of both the simple and complexed gels, under various wet and dry storage conditions, are shown in Fig. I. These clearly show the radical improvement of stability in this respect consequent on the complexing process. Measured sound speed in the gelatine-alginate complex (90.75% waterw/w) is 1519+ 1.2ms i at 20~C and variation with temperature in the range 1 7 . Y ' C - 2 5 ° C is linear with a coefficient of 2.6 ms-Z/~C. From a limited number of observations on the gel complex loaded with 120/~m polyethylene spheres up to concentration of 10 scatterers mm 3
there appears to be little dependence of sound speed on such loading. Corresponding results for graphite powder in gelatine gels have been reported by Madsen (1980) and in agar gels by Burlew et al. (1980). Figures 2 and 3 indicate the measured variations of attenuation coefficient with frequency observed both for the pure gel complex and for gels loaded with either 120 # m polyethylene spheres (Fig. 2) or 40/~m dia. particles of bovine peritoneal fat (Fig. 3). The corresponding data for a pure gelatine gel (90% water w/w) are also shown in Fig. 2 for comparison. DISCUSSION
The gelatine alginate gel provides a readily available and usable base material with properties that, combined with the introduction of scattering particles, allow for a whole variety of simple through to complex phantom constructions. The gel's ability to be stored under water, without need for elaborate protection, showing only minimal water uptake for periods of 6 months or longer, whilst maintaining its initial properties, is a considerable advantage over both simple and cross-linked gelatine gels. It is important, however, that the desirable properties of gelatine be maintained, and particularly the ease with which it enables manufacture of uniform, reproducible acoustic properties. So it is encouraging to note that a 10% gelatine gel, with comparable water levels to our new gel, also has very similar acoustic properties to the gel alginate material.
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Fig. 1. Measured weight change in 60 ml gelatine 16% wt./vol gel and gelatine-alginategel phantoms vs time stored under various conditions. Note that under "dry" conditions the weight-loss curve for gelatine-alginate complexis similar to that shown for pure gelatine./k, Gelatine immersedin water, 2ff~C. A, Gelatine immersedin water, 4°C. El, Gelatine plus 0.5% formaldehyde,immersed in water, 20°C. ~, Gelatine dry, 20"C. 0, Gelatine dry, 4°C. Gelatine-alginate immersed in water, 4°C. ©, Batch 1. Q, Batch 2.
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5 6 i 8 § lb Frequency(MHz) Fig. 2. Measured ultrasonic attenuation coefficient vs. frequency for pure and polyethylene loaded gelatine-alginate gels, at 22°C. E], Pure gelatine, 10%wt./vol, 90% waterw/w, O, pure gelatine-alginate. +, human liver (Bamber and Hill, 1979). Gelatine-alginate loaded with graded polyethylene 120/~m scatterers. i , 1666 scatterers cm -3. V, 3750 scatterers cm- 3. i,, 10000 scattererscm 3. O, 12500 scattererscm -3. Gelatine-alginate loaded with ungraded polyethylene l l9~um scatterers. A, 1666 scatterers cm 3. ~ , 3750 scatterers cm- 3. O
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W h e n studying the acoustic properties o f the new gel, it is i m p o r t a n t to note the variation o f properties as a function o f temperature. We measured this variation for velocity against temperature and, at 22°C, dc/dt = 2 . 6 m s - ~ / ° C : this m a y be c o m p a r e d with a value o f 1.8 m s - ~/°C for h u m a n liver tissue at 20°C as measured by Bamber and Hill (1979). D a t a for attenuation coefficient as a function o f scatterer concentrations and size, measured in gel p h a n t o m s using two different scatterer types (Figs. 2 and 3), as expected d e m o n s t r a t e d increased attenutation with higher scatterer concentrations. One interesting feature observed in these results was the effect o f scatterer size. The graded 120/am polyethylene particles produced a frequency related effect (evident as an inflection in the attenuation/frequency curve, irrespective o f concentration) at 4 M H z ; this feature was absent however in curves obtained either with the more heterogeneous polyethylene particles or with the bovine fat particles, which suggests that a frequency effect related to scatterer size is being d e m o n s t r a t e d with the graded particles. Clearly such an effect m a y need to be avoided for some applications and, with
Frequency(HHz) Fig. 3. Measured ultrasonic attenuation coefficient vs. frequency for gelatine-alginate gels loaded with various concentrations of bovine peritoneal fat (E particle dia. = 40/~m), at 22°C. O, Pure gelatine-alginate. Gelatine-atginate loaded with bovine peritoneal fat globules. O, 0.5% w/w. ~, 1%w/w./k, 2.5%w/w. [--], 5% w/w. O, 10% w/w.
A
B
C
D
*-3cm-4' Fig. 4. A B-scan image of a polyethylene loaded gel-alginate phantom. The phantom comprises four conical inserts embedded in a surrounding matrix, the polyethylene scatterer concentration in the matrix was 4/mm3and the inserts. (A) 4/mm 3. (B) 6/mm3. (C) 12/mm3. (D) 16/mm3. this proviso, the results presented here indicate that p h a n t o m s o f the type described can be m a d e to provide a g o o d m a t c h to h u m a n liver in terms o f frequency dependent attenuation coefficient.
Acknowledgements--Weare grateful to J. C. Bamber for help and advice with the attenuation measurements and to other colleagues for helpful discussions. The work was carried out with funds provided jointly by Medical Research Council and the Cancer Research Campaign.
484
N . L . BUSH and C. R. HILL REFERENCES
Astrahan M. A. (1979) Concerning hyperthermia phantom.Med. Phys. 6, 235. Bamber J. C., Fry M. J., Hill C. R. and Dunn F. (1977) Ultrasonic attenuation and backscattering by mammalian organs as a function of time after excision. Ultrasound in Med. & Biol. 3, 15-20. Bamber J. C. and Hill C. R. (1981) Acoustic properties of normal and cancerous human liver-l. Dependence on pathological condition. Ultrasound in Med. & Biol. 7, 121-133. Bamber J. C. and Hill C. R. (1979) Ultrasonic attenuation and propagation speed in mammalian tissues as a function of temperature. Ultrasound in Med. & Biol. 5, 149-157. Burlew M. M., Madsen E. L., Zagzebski J. A., Banjavic R. A. and Sum S. (1980) A new ultrasound tissue equivalent material. Radiology 134, 517-520. Edmonds P. D., Reyes Z., Filly R. A., Parkinson D. B. and Busey H. (1979) A human abdominal tissue phantom. In Ultrasonic tissue characterisation H(Edited by M. Linzer), pp. 323-326. NBS Spec. Publ. 525, Washington D. C. Eggleton R. C. and Whitcomb J. A. (1979) Tissue simulators for diagnostic ultrasound. In Ultrasonic Tissue Characterisation II (Edited by M. Linzer), pp. 327-336. NBS Spec. Publ. 525, U.S. Govt. Printing Office. Hall T. J., Bamber J. C. and Hill C. R. (1982) The influence of transducer characteristics and signal processing on fine structure in ultrasound images. Ultrasound in Med. Biol. 8 (Suppl. 1), 72. Le Gloahec V. C. R. (1947) Proteinous compound and the manufacture thereof. U.S. Pat. No. 2430, 180, 4 November. Madsen E. L., Zagzebski J. A., Banjavic R. A. and Jutila R. E. (1978) Tissue mimicking materials for ultrasound phantoms. Med. Phys. 5, 391-394. Madsen E. L. (1980) Ultrasonically soft-tissue mimicking materials.
In Medical Physics of CT and Ultrasound: Tissue imaging and characterisation (Edited by G. D. Fullerton and J. A. Zagzebski), pp. 531-550. American Institute of Physics, New York. Madsen E. L., Zagzebski J. A. and Frank G. R. (1982A) Oil in gelatin dispersions for use as ultrasonically tissue-mimicking materials. Ultrasound in Med. & Biol. 8, 277-287. Madsen E. L., Zagzebski J. A., Frank G. R., Greenleaf J. F. and Carson P. L. (1982b) Anthropomorphic breast phantoms for assessing ultrasonic imaging system performance and for training ultrasonographers--l. J. Clin. Ultrasound 10, 67-75.--II. J. Clin. Ultrasound 10, 91-100. Madsen E. L., Zagzebski J. A. and Ghilardi-Netto T. (1980) An anthropomorphic torso section phantom for ultrasonic imaging. Med. Phys. 7, 43-50. Madsen E. L., Zagzebski J. A. and Frank G. R. (1982d) An anthropomorphic ultrasound breast phantom containing intermediate-sized scatterers. Ultrasound in Med. & Biol. 8, 381 392. McDowell R. H. (1966) Alginates and their derivatives. Society of Chemical Industry, Monograph No. 24. The Chemistry and Rheology of water soluble gums and Colloids, pp. 19-32, SCI, 14 Belgrave Sq., London SWI. Merton J., Nicholas D. and Hill C. R. (1982) An assessment of the use of frequency and orientation diffraction for identifying scatterer concentration in models. Ultrasound in Med. & Biol. 8 (Suppl. 1), 128. Sommer F. G., Filly R. A., Edmonds P. D., Reyes Z. and Comas H. E. (1980) A phantom for imaging biological fluids by ultrasound and CT scanning. Ultrasound in Med. & Biol. 6, 135-140. tcr Haar G. and Carnochan P. (1982) A comparison of ultrasonic irradiation and RF inductive heating for clinical localised hyperthermia applications. Br. J. Cancer 45, Suppl. V, 77.