Reviewer's report 
 Grey scale enhancement by a new self-made contrast agent in early cirrhotic stage of rabbit liver Title: 
 1  19 September 2006 Version: Date: 
 Christoph F Dietrich Reviewer: 
Reviewer's report: 
General 
Review BioMed Gastroenterology 
Grey scale enhancement by a new self-made contrast agent in early cirrhotic stage of rabbit liver ' Li Zhang, Yun-You Duan, Ji-Kai Yin, Yong Zhang, Ji-Hong Cui and Tie-Sheng Cao BMC Gastroenterology Research article 
The authors describe the use of a new contrast agent in the evaluation of rabbit livers. 
General comments • Some misspellings should be corrected. 
• The authors should demonstrate why they did not use a conventional contrast agent instead of a self made 
one. The mention of a albumin perfluorocarbon ingredient of the new agent is not enough. The description 
should be given earlier in the methods section. Introduction 
- „So far, several UCAs, like Optison [3] and Definity [4] have been approved for treatment and become 
commercially available.“ (p2). The authors should mention for which treatment these agents are approved. 
To our knowledge they are approved for diagnosis. Methods 
1. The authors should value the clinical impact of biopsy in staging liver disease as it is not without failure 
when the liver presents with different disease stages. They should mention if there is a necessity for animal 
studies in evaluating contrast agents why they did not investigate the whole liver after the animals were 
killed. 
2. The authors should mention why there was no time intensity curve measurement of HA-HVTT, only for 
PIT. They discuss a difference of only 2 seconds of arrival time in HA and PV in the discussion section. 3. Statistical values should be given in the following manner: Mean value ? Standard deviaton [Minimum – Maximum] 
4. It should be discussed if it would be better to perform biopsy after CEUS since it is possible that hepatic 
perfusion is changed by liver trauma. 5. Where the investigators blinded? 6. Is there any financial interest in this new contrast agent? Are there any participations of pharmaceutical 
groups? 
7. In rabbits which hepatic vein was investigated? Is there a difference in hepatic vein arrival time after injection of a contrast agent? 8. In the introduction section they mention the potential of ultrasound contrast agents to produce Doppler 
shifts. Did they use a Doppler technique? In this case the MI of 0.24 is questionable high. Did they use a low 
MI harmonic technique (commercially available in Vivid 7?). The authors should discuss the technique used 
more precisely. 
9. “The self-made contrast agent (0.01mL/kg BW), was applied in randomized order into a marginal ear 
vein…“ (p3). What was randomised? 
Results The authors should add the information how long the contrast agent was detectable. Discussion 
“As we know, characterization of contrast agent is the….“ (p5). In this sentence characterisation is 
misleading due to the use in characterisation of liver tumours. The sentence should be rewritten „From the profile of time acoustic-intensity curve, we can comprehend the parenchymal enhancement phase in the liver followed the enhancement of the blood pool, the reason may be some of the Kuppfer 
cells take up the contrast agent.“ (p5). This should be confirmed by references. 
„While, in portal vein, microbubbles centralized easier than that in parenchyma“ (p5). The sentence is (a) incomplete and (b) what do the authors mean by centralized? Tables and Figures 
In figures the fibrotic stage of the rabbit liver should be added. 
Conclusion 
The paper is well written, has a satisfying style and describes the investigation eloquently and the topic is 
interesting. 
------------------------------------------------------------------------------- 
Major Compulsory Revisions (that the author must respond to before a decision on publication can be reached) 
Major weaknesses 
The authors should precise the advances in knowledge of this study. The authors do not precise why there is a necessity for animal studies in this subject. In staging liver disease there is a need for liver biopsy up to now, even if modern imaging methods can reveal substantial 
information which reflects in several medical guidelines. The subject of this study (staging liver fibrosis) has 
already been shown in patient’s studies and, therefore, an animal study should not be performed on this 
purpose. This study should be presented as a feasibility study for the new contrast agent and, therefore, the 
characteristics of this contrast agent including advantages and disadvantages in comparison to 
commercially available contrast agents which should be discussed precisely. In conclusion, the investigation itself has been discussed in a satisfactory way. The discussion section should be completely rewritten and the subject should actually be precised. Therefore, we suggest that this 
paper can be submitted again after substantial changes. 
------------------------------------------------------------------------------- 
Minor Essential Revisions (such as missing labels on figures, or the wrong use of a term, which the author 
can be trusted to correct) Minor weaknesses The paper lacks some information about the major use of contrast agents which is not mainly diffuse but 
focal liver disease. 
Furthermore, it would be appropriate to discuss more precisely the use of a new contrast agent. The authors state the size of the their bubbles is around 3-5 µm and would be larger than that of Optison and SonoVue?. The authors should 
- give references for the size of the mentioned commercially available agents. 
- state precisely why an agent consisting of albumin which is potentially dangerous (see also Optison?) should reveal advantages in comparison to low allergenic agents such as SonoVue? and Definity?. 
------------------------------------------------------------------------------- 
Discretionary Revisions (which the author can choose to ignore) 
 Unable to decide on acceptance or rejection until the authors have responded to the major What next?: 
compulsory revisions 
 An article of importance in its field Level of interest: 
 Acceptable Quality of written English: 
 No Statistical review: 
Declaration of competing interests: 
I declare that I have no competing interests 
Reviewer's report 
 Grey scale enhancement by a new self-made contrast agent in early cirrhotic stage of rabbit liver Title: 
 1  19 September 2006 Version: Date: 
 Christoph F Dietrich Reviewer: 
Reviewer's report: 
General 
Review BioMed Gastroenterology 
Grey scale enhancement by a new self-made contrast agent in early cirrhotic stage of rabbit liver ' Li Zhang, Yun-You Duan, Ji-Kai Yin, Yong Zhang, Ji-Hong Cui and Tie-Sheng Cao BMC Gastroenterology Research article 
The authors describe the use of a new contrast agent in the evaluation of rabbit livers. 
General comments • Some misspellings should be corrected. 
• The authors should demonstrate why they did not use a conventional contrast agent instead of a self made 
one. The mention of a albumin perfluorocarbon ingredient of the new agent is not enough. The description 
should be given earlier in the methods section. Introduction 
- „So far, several UCAs, like Optison [3] and Definity [4] have been approved for treatment and become 
commercially available.“ (p2). The authors should mention for which treatment these agents are approved. 
To our knowledge they are approved for diagnosis. Methods 
1. The authors should value the clinical impact of biopsy in staging liver disease as it is not without failure 
when the liver presents with different disease stages. They should mention if there is a necessity for animal 
studies in evaluating contrast agents why they did not investigate the whole liver after the animals were 
killed. 
2. The authors should mention why there was no time intensity curve measurement of HA-HVTT, only for 
PIT. They discuss a difference of only 2 seconds of arrival time in HA and PV in the discussion section. 3. Statistical values should be given in the following manner: Mean value ? Standard deviaton [Minimum – Maximum] 
4. It should be discussed if it would be better to perform biopsy after CEUS since it is possible that hepatic 
perfusion is changed by liver trauma. 5. Where the investigators blinded? 6. Is there any financial interest in this new contrast agent? Are there any participations of pharmaceutical 
groups? 
7. In rabbits which hepatic vein was investigated? Is there a difference in hepatic vein arrival time after injection of a contrast agent? 8. In the introduction section they mention the potential of ultrasound contrast agents to produce Doppler 
shifts. Did they use a Doppler technique? In this case the MI of 0.24 is questionable high. Did they use a low 
MI harmonic technique (commercially available in Vivid 7?). The authors should discuss the technique used 
more precisely. 
9. “The self-made contrast agent (0.01mL/kg BW), was applied in randomized order into a marginal ear 
vein…“ (p3). What was randomised? 
Results The authors should add the information how long the contrast agent was detectable. Discussion 
“As we know, characterization of contrast agent is the….“ (p5). In this sentence characterisation is 
misleading due to the use in characterisation of liver tumours. The sentence should be rewritten „From the profile of time acoustic-intensity curve, we can comprehend the parenchymal enhancement phase in the liver followed the enhancement of the blood pool, the reason may be some of the Kuppfer 
cells take up the contrast agent.“ (p5). This should be confirmed by references. 
„While, in portal vein, microbubbles centralized easier than that in parenchyma“ (p5). The sentence is (a) incomplete and (b) what do the authors mean by centralized? Tables and Figures 
In figures the fibrotic stage of the rabbit liver should be added. 
Conclusion 
The paper is well written, has a satisfying style and describes the investigation eloquently and the topic is 
interesting. 
------------------------------------------------------------------------------- 
Major Compulsory Revisions (that the author must respond to before a decision on publication can be reached) 
Major weaknesses 
The authors should precise the advances in knowledge of this study. The authors do not precise why there is a necessity for animal studies in this subject. In staging liver disease there is a need for liver biopsy up to now, even if modern imaging methods can reveal substantial 
information which reflects in several medical guidelines. The subject of this study (staging liver fibrosis) has 
already been shown in patient’s studies and, therefore, an animal study should not be performed on this 
purpose. This study should be presented as a feasibility study for the new contrast agent and, therefore, the 
characteristics of this contrast agent including advantages and disadvantages in comparison to 
commercially available contrast agents which should be discussed precisely. In conclusion, the investigation itself has been discussed in a satisfactory way. The discussion section should be completely rewritten and the subject should actually be precised. Therefore, we suggest that this 
paper can be submitted again after substantial changes. 
------------------------------------------------------------------------------- 
Minor Essential Revisions (such as missing labels on figures, or the wrong use of a term, which the author 
can be trusted to correct) Minor weaknesses The paper lacks some information about the major use of contrast agents which is not mainly diffuse but 
focal liver disease. 
Furthermore, it would be appropriate to discuss more precisely the use of a new contrast agent. The authors state the size of the their bubbles is around 3-5 µm and would be larger than that of Optison and SonoVue?. The authors should 
- give references for the size of the mentioned commercially available agents. 
- state precisely why an agent consisting of albumin which is potentially dangerous (see also Optison?) should reveal advantages in comparison to low allergenic agents such as SonoVue? and Definity?. 
------------------------------------------------------------------------------- 
Discretionary Revisions (which the author can choose to ignore) 
 Unable to decide on acceptance or rejection until the authors have responded to the major What next?: 
compulsory revisions 
 An article of importance in its field Level of interest: 
 Acceptable Quality of written English: 
 No Statistical review: 
Declaration of competing interests: 
I declare that I have no competing interests 
Please review this paper.
user
Reviewer's report 
 Grey scale enhancement by a new self-made contrast agent in early cirrhotic stage of rabbit liver Title: 
 1  19 September 2006 Version: Date: 
 Christoph F Dietrich Reviewer: 
Reviewer's report: 
General 
Review BioMed Gastroenterology 
Grey scale enhancement by a new self-made contrast agent in early cirrhotic stage of rabbit liver ' Li Zhang, Yun-You Duan, Ji-Kai Yin, Yong Zhang, Ji-Hong Cui and Tie-Sheng Cao BMC Gastroenterology Research article 
The authors describe the use of a new contrast agent in the evaluation of rabbit livers. 
General comments • Some misspellings should be corrected. 
• The authors should demonstrate why they did not use a conventional contrast agent instead of a self made 
one. The mention of a albumin perfluorocarbon ingredient of the new agent is not enough. The description 
should be given earlier in the methods section. Introduction 
- „So far, several UCAs, like Optison [3] and Definity [4] have been approved for treatment and become 
commercially available.“ (p2). The authors should mention for which treatment these agents are approved. 
To our knowledge they are approved for diagnosis. Methods 
1. The authors should value the clinical impact of biopsy in staging liver disease as it is not without failure 
when the liver presents with different disease stages. They should mention if there is a necessity for animal 
studies in evaluating contrast agents why they did not investigate the whole liver after the animals were 
killed. 
2. The authors should mention why there was no time intensity curve measurement of HA-HVTT, only for 
PIT. They discuss a difference of only 2 seconds of arrival time in HA and PV in the discussion section. 3. Statistical values should be given in the following manner: Mean value ? Standard deviaton [Minimum – Maximum] 
4. It should be discussed if it would be better to perform biopsy after CEUS since it is possible that hepatic 
perfusion is changed by liver trauma. 5. Where the investigators blinded? 6. Is there any financial interest in this new contrast agent? Are there any participations of pharmaceutical 
groups? 
7. In rabbits which hepatic vein was investigated? Is there a difference in hepatic vein arrival time after injection of a contrast agent? 8. In the introduction section they mention the potential of ultrasound contrast agents to produce Doppler 
shifts. Did they use a Doppler technique? In this case the MI of 0.24 is questionable high. Did they use a low 
MI harmonic technique (commercially available in Vivid 7?). The authors should discuss the technique used 
more precisely. 
9. “The self-made contrast agent (0.01mL/kg BW), was applied in randomized order into a marginal ear 
vein…“ (p3). What was randomised? 
Results The authors should add the information how long the contrast agent was detectable. Discussion 
“As we know, characterization of contrast agent is the….“ (p5). In this sentence characterisation is 
misleading due to the use in characterisation of liver tumours. The sentence should be rewritten „From the profile of time acoustic-intensity curve, we can comprehend the parenchymal enhancement phase in the liver followed the enhancement of the blood pool, the reason may be some of the Kuppfer 
cells take up the contrast agent.“ (p5). This should be confirmed by references. 
„While, in portal vein, microbubbles centralized easier than that in parenchyma“ (p5). The sentence is (a) incomplete and (b) what do the authors mean by centralized? Tables and Figures 
In figures the fibrotic stage of the rabbit liver should be added. 
Conclusion 
The paper is well written, has a satisfying style and describes the investigation eloquently and the topic is 
interesting. 
------------------------------------------------------------------------------- 
Major Compulsory Revisions (that the author must respond to before a decision on publication can be reached) 
Major weaknesses 
The authors should precise the advances in knowledge of this study. The authors do not precise why there is a necessity for animal studies in this subject. In staging liver disease there is a need for liver biopsy up to now, even if modern imaging methods can reveal substantial 
information which reflects in several medical guidelines. The subject of this study (staging liver fibrosis) has 
already been shown in patient’s studies and, therefore, an animal study should not be performed on this 
purpose. This study should be presented as a feasibility study for the new contrast agent and, therefore, the 
characteristics of this contrast agent including advantages and disadvantages in comparison to 
commercially available contrast agents which should be discussed precisely. In conclusion, the investigation itself has been discussed in a satisfactory way. The discussion section should be completely rewritten and the subject should actually be precised. Therefore, we suggest that this 
paper can be submitted again after substantial changes. 
------------------------------------------------------------------------------- 
Minor Essential Revisions (such as missing labels on figures, or the wrong use of a term, which the author 
can be trusted to correct) Minor weaknesses The paper lacks some information about the major use of contrast agents which is not mainly diffuse but 
focal liver disease. 
Furthermore, it would be appropriate to discuss more precisely the use of a new contrast agent. The authors state the size of the their bubbles is around 3-5 µm and would be larger than that of Optison and SonoVue?. The authors should 
- give references for the size of the mentioned commercially available agents. 
- state precisely why an agent consisting of albumin which is potentially dangerous (see also Optison?) should reveal advantages in comparison to low allergenic agents such as SonoVue? and Definity?. 
------------------------------------------------------------------------------- 
Discretionary Revisions (which the author can choose to ignore) 
 Unable to decide on acceptance or rejection until the authors have responded to the major What next?: 
compulsory revisions 
 An article of importance in its field Level of interest: 
 Acceptable Quality of written English: 
 No Statistical review: 
Declaration of competing interests: 
I declare that I have no competing interests 
assistant