HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Figures Only Full Text (PDF) All Versions of this Article: 2313030544v1 231/3/785    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lim, A. K. P. Articles by Blomley, M. J. K. Search for Related Content PubMed PubMed Citation Articles by Lim, A. K. P. Articles by Blomley, M. J. K.

Evidence for Spleen-specific Uptake of a Microbubble Contrast Agent: A Quantitative Study in Healthy Volunteers¹

¹ From the Imaging Sciences Department, MRC Clinical Sciences Centre (A.K.P.L., N.P., R.J.E., S.D.T.R., D.O.C., M.J.K.B.) and Department of Medicine A, Faculty of Medicine (N.P., S.D.T.R.), Imperial College London, Hammersmith Hospital, DuCane Rd, London W12 0HS, England. Received April 7, 2003; revision requested June 24; final revision received November 6; accepted November 20. Supported by Toshiba, Tokyo, Japan, and Bracco, Milan, Italy. A.K.P.L. supported by the United Kingdom National Health Service Research and Development Initiative and the Kodak Scholarship, Royal College of Radiologists. Address correspondence to A.K.P.L. (e-mail: a.lim@imperial.ac.uk).

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: To evaluate the pharmacokinetics of the microbubble contrast agent BR1.

MATERIALS AND METHODS: Twenty healthy volunteers were injected via arm vein with a 1.2-mL bolus of BR1. Ultrasonographic images of liver and right kidney and of spleen and left kidney were obtained intermittently for 5 minutes with low-mechanical-index software (to minimize microbubble destruction) that shows stationary microbubbles in green. Percentage total uptake was calculated as the number of green pixels in the region of interest for each organ over time, divided by the total pixels. Relative uptake, the ratio of total uptake in liver to that in right kidney and of total uptake in spleen to that in left kidney, and differential uptake, the difference in total uptake between liver and right kidney and between spleen and left kidney, were calculated. Total uptake for each organ was plotted against time, and the gradient of a best-fit straight line was calculated. Wilcoxon signed rank test was used to compare mean uptake values in each subject. Mann-Whitney U test was used for comparisons in sex and age.

RESULTS: Total uptake declined over 5 minutes in left and right kidney and in liver (from 88% ± 10% [1 minute] to 67% ± 14% [5 minutes]), but not in spleen (range, 90%–99%). Mean relative uptake ± 1 SD for spleen increased from 2.3 ± 0.7 (1 minute) to 3.7 ± 2.3 (5 minutes) (P = .005) but for liver was constant: 2.1 ± 0.9 (1 minute) and 2.3 ± 0.4 (5 minutes) (P = .06). Mean differential uptake ± 1 SD for spleen increased from 51.3% ± 14.9% (1 minute) to 65.0% ± 9.1% (5 minutes) (P = .002). Significant difference was seen over time in total uptake gradients between spleen and left kidney (P = .014) but not between liver and right kidney or right and left kidney. No difference was seen between men and women or with age.

CONCLUSION: BR1 produces spleen-specific enhancement that is longer (5 minutes) than the blood pool phase.

© RSNA, 2004

Index terms: Kidney, US, 81.12988 • Liver, US, 761.12988 • Microbubbles • Spleen, US, 775.12988 • Ultrasound (US), contrast media

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The majority of microbubble agents were initially thought to be blood pool agents. However, some have recently been shown to exhibit specific hepatosplenic uptake after their disappearance from the blood pool. These include SH U 508A (Levovist; Schering, Berlin, Germany) (1), SH U 563A (Sonovist; Schering) (2), NC100100 (Sonazoid; Nycomed Amersham, Oslo, Norway) (3), and BR14 (Bracco, Milan, Italy) (4). The site of accumulation for these microbubble agents is unknown, but it may be within the reticuloendothelial system or sinusoid (1–7). Sonovist has been shown to be phagocytosed by Kupffer cells (3), presumably because of its very stable outer shell, while Levovist has only a surfactant outer surface and is unlikely to be stable enough to be taken up by the reticuloendothelial system. Marked splenic tropism has not been described for any of these agents.

One of the recently introduced microbubble agents, BR1 (SonoVue; Bracco), is based on a perfluoro gas and sulfur hexafluoride, with a phospholipid membrane. Stability and antiaggregation properties are also provided by the addition of several surfactants (polyethylene glycol and palmitic acid). BR1 was thought to be purely a vascular agent with no tissue tropism (7,8). While performing clinical studies with this agent, we have observed marked splenic uptake. Thus, the purpose of our study was to evaluate the pharmacokinetics of the microbubble contrast agent BR1.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Subjects
Twenty healthy volunteers (nine men, 11 women) with a mean age of 40 years in men (range, 27–57 years), 39 years in women (range, 23–48 years), and an overall mean age of 39.3 years (range, 23–57 years) were studied. Informed consent was obtained, as well as approval from the Ethics Committees of the Hammersmith Hospitals NHS Trust, London (REC 2002/6271). Each volunteer was asked to include any important medical history—in particular, liver, splenic, or renal disease. Individuals who were following a regular medication regimen or had a focal lesion in any of the aforementioned organs at baseline ultrasonography (US) were excluded from the study.

Each subject fasted for a minimum of 6 hours and was then administered a single bolus injection (1.2 mL) of the microbubble agent BR1 (SonoVue; Bracco) via an antecubital vein. US (Aplio; Toshiba, Tokyo, Japan) images of the liver and right kidney and of the spleen and left kidney were obtained intermittently (every 30 seconds) by using low-mechanical-index software (Vascular Recognition Imaging; Toshiba) that minimized microbubble destruction and is a standardized package on all Aplio machines. All images were obtained by the same experienced sonologist (N.P.).

The low-mechanical-index scanning software that we used allows the microbubble and gray-scale information to be collected separately by using a color overlay to display microbubble data. Stationary microbubbles within tissue are depicted in green, and mobile microbubbles within vessels are in either red or blue, depending on the direction of flow. Microbubble localization within organ tissue can thus be depicted by counting the number of green pixels.

The images were collected between 1 and 5 minutes after injection of BR1 at approximately 30-second intervals (± 15 seconds) with constant scanner settings. Initially, the depth was altered, according to the habitus of the volunteer, to maximize the view of either the liver and right kidney or the spleen and left kidney in the longitudinal section. The scanner parameters were then unaltered during the 5 minutes of data acquisition. With the low-mechanical-index scanning mode we used, we maintained the same mechanical index (acoustic power, 0.4%) for all subjects, as suggested by the manufacturer, to minimize bubble destruction. We also avoided the inclusion of any large vessels on the images obtained for analysis. The digital images were then transferred to a personal computer via optical disks.

Quantitation
All quantitation was performed by N.P., by using the Color Quantification software package (Kinetic Imaging, Nottingham, England). Regions of interest (on average, 2,000–2,500 pixels) were drawn for each organ at approximately the same depth on every image—liver and right kidney or spleen and left kidney (Fig 1). The background gray-scale image with color overlay was used to ensure that regions of interest were accurately drawn over the relevant organs at the same depth. The green pixel count was then performed with the gray-scale component removed.

View larger version (76K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Longitudinal US image shows the placement of regions of interest (circles) at approximately the same depth in the spleen and left kidney in a volunteer after injection of 1.2 mL of BR1. The amount of BR1 in each organ at a particular time was calculated as the number of green pixels in the designated region, expressed as a percentage of the total number of pixels and was termed total uptake.

The relative uptake was also calculated as the ratios of the total uptake in the liver to that in the right kidney and of total uptake in the spleen to that in the left kidney. The differential uptake was defined as the difference between the total uptake in the liver and that in the right kidney and between that in the spleen and that in the left kidney. Both the relative and differential uptake were calculated at 1 minute (± 15 seconds) and at 5 minutes (± 15 seconds) after injection.

The total uptake for each organ in each volunteer was plotted against time, a best-fit straight line was drawn, and gradients were calculated. The mean gradients over time for each organ in each volunteer were then analyzed.

Statistical Analysis
The mean relative uptake at 1 minute and at 5 minutes for all patients, as well as the mean relative gradients (ie, liver to right kidney and spleen to left kidney), were compared by using a nonparametric paired sample test, the Wilcoxon signed rank test, with a statistical software program (SPSS version 10.1; SPSS, Chicago, Ill). The Mann-Whitney U test was used to compare the mean gradients of total uptake, relative uptake, and differential uptake for each organ between men and women and between different ages by using the same statistical program. A P value of less than .05 was considered to indicate a significant difference.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The images in Figure 2 illustrate a typical series obtained in the liver and right kidney and the spleen and left kidney in the same volunteer over time after injection with the evaluated microbubble contrast agent.

View larger version (96K): [in this window] [in a new window] [Download PPT slide]   Figure 2a. Longitudinal US images in one volunteer over time illustrate a typical series obtained with a low-power nondisruptive mode, in which stationary microbubbles are depicted in green, and microbubbles within vessels are shown as red or blue, depending on direction of flow. (a-c) Images of liver and right kidney obtained at (a) baseline, (b) 90 seconds after injection with BR1 (1.2 mL), and (c) 5 minutes after injection. Note decreased number of green pixels in the liver and right kidney at 5 minutes. (d-f) Comparative images of spleen and left kidney obtained at (d) baseline, (e) 75 seconds after injection with BR1 (1.2 mL), and (f) 5 minutes after injection. Note that the number of green pixels in the spleen at 5 minutes appears similar to that at 75 seconds.

View larger version (79K): [in this window] [in a new window] [Download PPT slide]   Figure 2b. Longitudinal US images in one volunteer over time illustrate a typical series obtained with a low-power nondisruptive mode, in which stationary microbubbles are depicted in green, and microbubbles within vessels are shown as red or blue, depending on direction of flow. (a-c) Images of liver and right kidney obtained at (a) baseline, (b) 90 seconds after injection with BR1 (1.2 mL), and (c) 5 minutes after injection. Note decreased number of green pixels in the liver and right kidney at 5 minutes. (d-f) Comparative images of spleen and left kidney obtained at (d) baseline, (e) 75 seconds after injection with BR1 (1.2 mL), and (f) 5 minutes after injection. Note that the number of green pixels in the spleen at 5 minutes appears similar to that at 75 seconds.

View larger version (100K): [in this window] [in a new window] [Download PPT slide]   Figure 2c. Longitudinal US images in one volunteer over time illustrate a typical series obtained with a low-power nondisruptive mode, in which stationary microbubbles are depicted in green, and microbubbles within vessels are shown as red or blue, depending on direction of flow. (a-c) Images of liver and right kidney obtained at (a) baseline, (b) 90 seconds after injection with BR1 (1.2 mL), and (c) 5 minutes after injection. Note decreased number of green pixels in the liver and right kidney at 5 minutes. (d-f) Comparative images of spleen and left kidney obtained at (d) baseline, (e) 75 seconds after injection with BR1 (1.2 mL), and (f) 5 minutes after injection. Note that the number of green pixels in the spleen at 5 minutes appears similar to that at 75 seconds.

View larger version (89K): [in this window] [in a new window] [Download PPT slide]   Figure 2d. Longitudinal US images in one volunteer over time illustrate a typical series obtained with a low-power nondisruptive mode, in which stationary microbubbles are depicted in green, and microbubbles within vessels are shown as red or blue, depending on direction of flow. (a-c) Images of liver and right kidney obtained at (a) baseline, (b) 90 seconds after injection with BR1 (1.2 mL), and (c) 5 minutes after injection. Note decreased number of green pixels in the liver and right kidney at 5 minutes. (d-f) Comparative images of spleen and left kidney obtained at (d) baseline, (e) 75 seconds after injection with BR1 (1.2 mL), and (f) 5 minutes after injection. Note that the number of green pixels in the spleen at 5 minutes appears similar to that at 75 seconds.

View larger version (69K): [in this window] [in a new window] [Download PPT slide]   Figure 2e. Longitudinal US images in one volunteer over time illustrate a typical series obtained with a low-power nondisruptive mode, in which stationary microbubbles are depicted in green, and microbubbles within vessels are shown as red or blue, depending on direction of flow. (a-c) Images of liver and right kidney obtained at (a) baseline, (b) 90 seconds after injection with BR1 (1.2 mL), and (c) 5 minutes after injection. Note decreased number of green pixels in the liver and right kidney at 5 minutes. (d-f) Comparative images of spleen and left kidney obtained at (d) baseline, (e) 75 seconds after injection with BR1 (1.2 mL), and (f) 5 minutes after injection. Note that the number of green pixels in the spleen at 5 minutes appears similar to that at 75 seconds.

View larger version (70K): [in this window] [in a new window] [Download PPT slide]   Figure 2f. Longitudinal US images in one volunteer over time illustrate a typical series obtained with a low-power nondisruptive mode, in which stationary microbubbles are depicted in green, and microbubbles within vessels are shown as red or blue, depending on direction of flow. (a-c) Images of liver and right kidney obtained at (a) baseline, (b) 90 seconds after injection with BR1 (1.2 mL), and (c) 5 minutes after injection. Note decreased number of green pixels in the liver and right kidney at 5 minutes. (d-f) Comparative images of spleen and left kidney obtained at (d) baseline, (e) 75 seconds after injection with BR1 (1.2 mL), and (f) 5 minutes after injection. Note that the number of green pixels in the spleen at 5 minutes appears similar to that at 75 seconds.

Differential Uptake
The findings for differential uptake were similar to those for relative uptake. The mean differential uptake ± 1 SD for the spleen increased from 51.3% ± 14.9% at 1 minute to 65.0% ± 9.1% at 5 minutes (Wilcoxon signed rank test, P = .002), while the differential uptake for the liver remained constant: 36.0% ± 13.9% at 1 minute and 40.0% ± 14.2% at 5 minutes (Wilcoxon signed rank test, P = .56).

A significant difference was also observed between the gradients of the total uptake over time in the spleen compared with the left kidney (P = .014), but no difference was seen between the liver and right kidney or between the right and left kidneys. The mean gradients of each organ are shown in Table 3, and the data are also illustrated in a graph in Figure 3, in which the difference between the spleen and other organs can be more easily observed.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Graph shows mean total uptake against time (seconds) in all the volunteers for the spleen (), liver (), and right () and left () kidneys. Gradients of best-fit straight lines are similar for the liver and both kidneys. However, gradient for the spleen is virtually a horizontal line, with all values 90%-99%.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
At present, there are only four microbubble agents with recognized hepatosplenic uptake. These include SH U 508A (Levovist) (1), SH U 563A (Sonovist) (2), NC100100 (Sonazoid) (3), and BR14 (4). The most widely used microbubble in Europe, BR1 (SonoVue), has always been thought to be a purely vascular contrast agent (7,8).

By using the quantitation technique of counting the number of green pixels within a region of interest in an organ at a particular time, with the assumption that these were proportional to the number of stationary microbubbles within the organ parenchyma during scanning with the low-mechanical-index mode, we have shown that BR1 is selectively taken up by the spleen 5 minutes past the blood pool phase. As shown in Figure 3, the total uptake in the spleen was constant over 5 minutes, while there was a reduction of microbubble concentration within the liver and both kidneys during this time. This observation is also supported by the results of analysis of relative uptake and differential uptake, in which differences were seen only for the spleen. We believe this is the first time that specificity for splenic tissue has been described for this microbubble contrast agent. One limitation of our study is that it did not address how long this effect persisted within the spleen after the 5-minute data collection period.

The results showed that, although there was a greater total uptake in the liver compared with that in the right kidney, there was decay of the microbubble contrast agent within the parenchyma at a similar rate for the liver and both kidneys. Thus, the higher total uptake values in the liver for all volunteers seem to reflect the larger vascular volume of the liver in comparison with that of the kidneys. These data suggest that, within the liver, this microbubble contrast agent is purely a vascular agent, with no evidence of selective uptake.

Our quantitation of selective uptake was reliant to some extent on the software, including the assumption that the low-mechanical-index mode used in this study accurately depicts stationary microbubbles, as indicated by the manufacturer. There may have been microbubbles moving very slowly on our images that were depicted in green, as though they were stationary, which might have led to small errors in quantitation; however, we attempted to minimize this effect by trying to ensure the same image plane and scanning parameters over time and by avoiding large vessels.

Spleen specificity, to the best of our knowledge, has not previously been attributed to this contrast agent. Further phase microscopic studies similar to those performed by Iijima and colleagues (9) by using in vitro Kupffer cells are required to ascertain precisely where these microbubbles are within the spleen. Are they phagocytosed by macrophages or merely trapped within the splenic parenchyma? It is also possible that the behavior of BR1 could be analogous to that of the heat-damaged red blood cells at nuclear imaging, which are highly spleen specific and are not taken up by the liver, unlike colloid-labeled tracers (10,11). If the latter is true, then BR1 could provide a useful alternative in the identification or confirmation of splenic tissue, as well as in characterization of focal splenic lesions. It would also aid in detecting damage to the spleen in patients with abdominal trauma.

The mechanism for hepatosplenic uptake of microbubble agents remains unclear and contentious, even though four microbubble agents have previously been recognized to have properties for hepatosplenic uptake (1–4). The specific tropism of the microbubble contrast agent BR1 to the spleen without hepatic uptake is unusual, and the results of our study provide a small step toward understanding the kinetics of US microbubble contrast agents, which otherwise at present remain poorly understood.

In conclusion, we have shown that BR1 produces a spleen-specific enhancement that lasts longer (5 minutes) than the blood pool and liver enhancement phases. This unique splenic specificity would be useful for the detection of splenic tissue and characterization of focal lesions.

	ACKNOWLEDGMENTS

 
We thank all those who kindly volunteered to take part in this study.

	FOOTNOTES

 
Author contributions: Guarantors of integrity of entire study, A.K.P.L., M.J.K.B., N.P.; study concepts, A.K.P.L., M.J.K.B., D.O.C.; study design, A.K.P.L., M.J.K.B., R.J.E.; literature research, A.K.P.L.; clinical studies, A.K.P.L., N.P., M.J.K.B.; data acquisition, A.K.P.L., N.P., R.J.E.; data analysis/interpretation, A.K.P.L., N.P., M.J.K.B., R.J.E.; statistical analysis, A.K.P.L.; manuscript preparation and editing, A.K.P.L.; manuscript definition of intellectual content, A.K.P.L., M.J.K.B., S.D.T.R., D.O.C.; manuscript revision/review and final version approval, all authors

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Blomley MJ, Albrecht T, Cosgrove DO, et al. Stimulated acoustic emission to image a late liver and spleen-specific phase of Levovist in normal volunteers and patients with and without liver disease. Ultrasound Med Biol 1999; 25:1341-1352.[CrossRef][Medline]
2. Forsberg F, Goldberg BB, Liu JB, et al. Tissue-specific US contrast agent for evaluation of hepatic and splenic parenchyma. Radiology 1999; 210:125-132.[Abstract/Free Full Text]
3. Leen E, Ramnarine K, Kyriakopoulou K, et al. Improved characterization of focal liver tumors: dynamic Doppler imaging using NC100100—a new liver specific echo-enhancer (abstr). Radiology 1998; 209(P):293.
4. Schneider M, Broillet A, Bussat P, et al. Gray-scale liver enhancement in VX2 tumor-bearing rabbits using BR14, a new ultrasonographic contrast agent. Invest Radiol 1997; 32:410-417.[CrossRef][Medline]
5. Kono Y, Steinbach GC, Peterson T, Schmid-Schonbein GW, Mattrey RF. Mechanism of parenchymal enhancement of the liver with a microbubble-based US contrast medium: an intravital microscopy study in rats. Radiology 2002; 224:253-257.[Abstract/Free Full Text]
6. Schneider M, Arditi M, Barrau MB, et al. BR1: a new ultrasonographic contrast agent based on sulfur hexafluoride-filled microbubbles. Invest Radiol 1995; 30:451-457.[Medline]
7. Harvey CJ, Blomley MJ, Eckersley RJ, Cosgrove DO. Developments in ultrasound contrast media. Eur Radiol 2001; 11:675-689.[CrossRef][Medline]
8. Correas JM, Bridal L, Lesavre A, Mejean A, Claudon M, Helenon O. Ultrasound contrast agents: properties, principles of action, tolerance, and artifacts. Eur Radiol 2001; 11:1316-1328.[CrossRef][Medline]
9. Iijima H, Miyahara T, Suzuki S, et al. Sinusoidal endothelium and microbubble: Kupffer imaging and bioeffect (abstr). Ultrasound Med Biol 2003; 29:S222.
10. Person RE, Bender JM. Hepatic lesion differentiated from accessory spleen by a heat-damaged red blood cell scan. Clin Nucl Med 2000; 25:516-518.[CrossRef][Medline]
11. Massey MD, Stevens JS. Residual spleen found on denatured red blood cell scan following negative colloid scans. J Nucl Med 1991; 32:2286-2287.[Abstract/Free Full Text]

This article has been cited by other articles:

				M. Picardi, A. Soricelli, F. Pane, P. Zeppa, E. Nicolai, M. De Laurentiis, F. Grimaldi, and B. Rotoli Contrast-enhanced Harmonic Compound US of the Spleen to Increase Staging Accuracy in Patients with Hodgkin Lymphoma: A Prospective Study Radiology, May 1, 2009; 251(2): 574 - 582. [Abstract] [Full Text] [PDF]

				C. Gorg, R. Kring, and T. Bert Transcutaneous Contrast-Enhanced Sonography of Peripheral Lung Lesions Am. J. Roentgenol., October 1, 2006; 187(4): W420 - W429. [Abstract] [Full Text] [PDF]

				A. K. P. Lim, N. Patel, R. J. Eckersley, R. D. Goldin, H. C. Thomas, D. O. Cosgrove, S. D. Taylor-Robinson, and M. J. K. Blomley Hepatic Vein Transit Time of SonoVue: A Comparative Study with Levovist Radiology, July 1, 2006; 240(1): 130 - 135. [Abstract] [Full Text] [PDF]

				C. Gorg, C. Graef, and T. Bert Contrast-Enhanced Sonography for Differential Diagnosis of an Inhomogeneous Spleen of Unknown Cause in Patients With Pain in the Left Upper Quadrant J. Ultrasound Med., June 1, 2006; 25(6): 729 - 734. [Abstract] [Full Text] [PDF]

				V R Stewart and P S Sidhu New directions in ultrasound: microbubble contrast. Br. J. Radiol., March 1, 2006; 79(939): 188 - 194. [Full Text] [PDF]

				C. Gorg and T. Bert Second-generation sonographic contrast agent for differential diagnosis of perisplenic lesions. Am. J. Roentgenol., March 1, 2006; 186(3): 621 - 626. [Abstract] [Full Text] [PDF]

				H Maruyama, S Matsutani, H Saisho, Y Mine, N Kamiyama, T Hirata, and M Sasamata Real-time blood-pool images of contrast enhanced ultrasound with Definity in the detection of tumour nodules in the liver Br. J. Radiol., June 1, 2005; 78(930): 512 - 518. [Abstract] [Full Text] [PDF]

				O. Catalano, F. Sandomenico, I. Matarazzo, and A. Siani Contrast-Enhanced Sonography of the Spleen Am. J. Roentgenol., April 1, 2005; 184(4): 1150 - 1156. [Abstract] [Full Text] [PDF]

This Article Abstract Figures Only Full Text (PDF) All Versions of this Article: 2313030544v1 231/3/785    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lim, A. K. P. Articles by Blomley, M. J. K. Search for Related Content PubMed PubMed Citation Articles by Lim, A. K. P. Articles by Blomley, M. J. K.