Reviewer's report 
 Diagnostic Value of Biochemical Markers (NashTest) for the prediction of NASH in Patients with Title: 
Non-Alcoholic Fatty Liver Disease 
 1  9 September 2006 Version: Date: 
 David Kleiner Reviewer: 
Reviewer's report: 
General 
This paper describes the use of a proprietary combination of biochemical assays and demographic/physical 
characteristics to predict whether or not a patient’s liver biopsy will show steatohepatitis. 
------------------------------------------------------------------------------- 
Major Compulsory Revisions (that the author must respond to before a decision on publication can be 
reached) 
1. Methods, p.10, paragraph beginning “Steatohepatitis was defined as a NAS score of 5 or greater.” In this 
study, the diagnosis of NASH is based on the numerical outcome of a recently developed scoring system 
for NASH (ref #14)—in particular the NASH Activity Score (NAS). The authors categorize the fatty liver 
disease based on the value of the NAS, with 0-2 being not diagnostic of NASH, 3-4 being borderline NASH and 5-8 being diagnostic of NASH. This is a misinterpretation of the method. In fact, the data in reference 14 does not support diagnosis by numbers and the authors of that paper note that the NAS is intended for 
use in monitoring changes in liver disease and other clinical situations, and is not intended to replace the 
pathologist’s determination of whether NASH is present or not. The use of the scoring system as proposed 
in ref. 14 is entirely in line with the use of scoring systems in other liver diseases, where scores are used to scale severity and not to determine whether or not the patient has the liver disease under study. To the 
extent that the NAS represents the severity of current liver injury, the proposed NashTest may separate 
those with more severe injury from those with little injury. This would be of great value in clinical trial situations where the investigator might want to enroll those with severe disease first or perhaps for 
identifying patients at greatest risk for progression. However, given that about a third of biopsies in the NAS 
3-4 range in ref. 14 were definite NASH and another third not diagnostic of NASH, the utility of the 
NashTest for identifying patients with bona fide NASH is questionable. The authors need to diagnostically categorize the biopsies according to accepted criteria (which may be found in many publications), independent of the NAS and proceed on that basis or revise the article and test as one which looks at 
severity of injury and does not make claims about diagnosis. 
2. Methods, p.11, paragraph beginning “The new panel, NT…”. There is almost no detail about how the 
base data was combined to give NT. Given that this is intended to be the critical paper that validates the use 
of the assay this is too little detail. The authors have a patent pending on the methodology of deriving the 
NashTest—it is not unreasonable for them to share more of the basic assay and calculation information with 
the readers. The authors need to provide more information on the derivation of this test and show the 
appropriate supporting data. 3. Results, general comment. This comment is related to #2 above. There is no data related to the 
distribution of the NashTest values over the various diagnostic categories or any of the performance 
characteristics of the assay. Leaving aside the issue of whether the cases are grouped appropriately for diagnosis, these are critical data completely missing from this paper. The test is presented as a black box that generates diagnoses. The authors would like to demonstrate their new test as a way to diagnose liver 
disease but offer no data (other than the categorization by the test) on the test itself. Data on the test itself 
needs to be shown. 
------------------------------------------------------------------------------- 
Minor Essential Revisions (such as missing labels on figures, or the wrong use of a term, which the author 
can be trusted to correct) 
4. Methods, p.9 “All patients and controls gave informed consent for the use of data and serum for research 
purposes.” No mention is made of approval of the use of the biopsy for research purposes; this should be 
noted as well along with any institutional oversight of this project. 
5. Results, data presentation. The authors present all of their data in summary tables as means, standard errors and p values. This type of presentation would be enhanced by figures showing the distribution of critical data by category. Box plots are very useful for this purpose and since there are no limitations on 
space for this journal, I would recommend that the authors show as much of the critical data as possible. 
6. Results, Table 1. Fibrosis Stage. The use of F0, F1 etc. to denote fibrosis stage is a peculiarity of the 
Metavir scoring system and should not be used for general designation of fibrosis stage in other systems. 
7. Figure 1C (presumably) is missing. 
------------------------------------------------------------------------------- 
Discretionary Revisions (which the author can choose to ignore) 8. Introduction, p.5 “…there is considerable sampling variability; and high intra- and inter-pathologist 
variability…”. This is a subjective value judgment for which there is no data (despite the references). There 
is no more variability in determination of the diagnosis of NASH than in many other medical diseases that lack highly specific and sensitive tests and the variability between observers is less than that of many diseases and clinical judgments. If a clinical serum test is good enough to replace a tissue biopsy and 
pathologic examination, then it will, no matter how good the biopsy is. There are many historical examples 
in which tissue pathology has been replaced by other kinds of tests and it was not necessary to make such arguments against the use of the biopsy. I would suggest that these sorts of pejorative comments be removed from the introduction and discussion sections. They really don’t add to the argument for using 
biomarkers or serum tests. 
 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 
 Diagnostic Value of Biochemical Markers (NashTest) for the prediction of NASH in Patients with Title: 
Non-Alcoholic Fatty Liver Disease 
 1  9 September 2006 Version: Date: 
 David Kleiner Reviewer: 
Reviewer's report: 
General 
This paper describes the use of a proprietary combination of biochemical assays and demographic/physical 
characteristics to predict whether or not a patient’s liver biopsy will show steatohepatitis. 
------------------------------------------------------------------------------- 
Major Compulsory Revisions (that the author must respond to before a decision on publication can be 
reached) 
1. Methods, p.10, paragraph beginning “Steatohepatitis was defined as a NAS score of 5 or greater.” In this 
study, the diagnosis of NASH is based on the numerical outcome of a recently developed scoring system 
for NASH (ref #14)—in particular the NASH Activity Score (NAS). The authors categorize the fatty liver 
disease based on the value of the NAS, with 0-2 being not diagnostic of NASH, 3-4 being borderline NASH and 5-8 being diagnostic of NASH. This is a misinterpretation of the method. In fact, the data in reference 14 does not support diagnosis by numbers and the authors of that paper note that the NAS is intended for 
use in monitoring changes in liver disease and other clinical situations, and is not intended to replace the 
pathologist’s determination of whether NASH is present or not. The use of the scoring system as proposed 
in ref. 14 is entirely in line with the use of scoring systems in other liver diseases, where scores are used to scale severity and not to determine whether or not the patient has the liver disease under study. To the 
extent that the NAS represents the severity of current liver injury, the proposed NashTest may separate 
those with more severe injury from those with little injury. This would be of great value in clinical trial situations where the investigator might want to enroll those with severe disease first or perhaps for 
identifying patients at greatest risk for progression. However, given that about a third of biopsies in the NAS 
3-4 range in ref. 14 were definite NASH and another third not diagnostic of NASH, the utility of the 
NashTest for identifying patients with bona fide NASH is questionable. The authors need to diagnostically categorize the biopsies according to accepted criteria (which may be found in many publications), independent of the NAS and proceed on that basis or revise the article and test as one which looks at 
severity of injury and does not make claims about diagnosis. 
2. Methods, p.11, paragraph beginning “The new panel, NT…”. There is almost no detail about how the 
base data was combined to give NT. Given that this is intended to be the critical paper that validates the use 
of the assay this is too little detail. The authors have a patent pending on the methodology of deriving the 
NashTest—it is not unreasonable for them to share more of the basic assay and calculation information with 
the readers. The authors need to provide more information on the derivation of this test and show the 
appropriate supporting data. 3. Results, general comment. This comment is related to #2 above. There is no data related to the 
distribution of the NashTest values over the various diagnostic categories or any of the performance 
characteristics of the assay. Leaving aside the issue of whether the cases are grouped appropriately for diagnosis, these are critical data completely missing from this paper. The test is presented as a black box that generates diagnoses. The authors would like to demonstrate their new test as a way to diagnose liver 
disease but offer no data (other than the categorization by the test) on the test itself. Data on the test itself 
needs to be shown. 
------------------------------------------------------------------------------- 
Minor Essential Revisions (such as missing labels on figures, or the wrong use of a term, which the author 
can be trusted to correct) 
4. Methods, p.9 “All patients and controls gave informed consent for the use of data and serum for research 
purposes.” No mention is made of approval of the use of the biopsy for research purposes; this should be 
noted as well along with any institutional oversight of this project. 
5. Results, data presentation. The authors present all of their data in summary tables as means, standard errors and p values. This type of presentation would be enhanced by figures showing the distribution of critical data by category. Box plots are very useful for this purpose and since there are no limitations on 
space for this journal, I would recommend that the authors show as much of the critical data as possible. 
6. Results, Table 1. Fibrosis Stage. The use of F0, F1 etc. to denote fibrosis stage is a peculiarity of the 
Metavir scoring system and should not be used for general designation of fibrosis stage in other systems. 
7. Figure 1C (presumably) is missing. 
------------------------------------------------------------------------------- 
Discretionary Revisions (which the author can choose to ignore) 8. Introduction, p.5 “…there is considerable sampling variability; and high intra- and inter-pathologist 
variability…”. This is a subjective value judgment for which there is no data (despite the references). There 
is no more variability in determination of the diagnosis of NASH than in many other medical diseases that lack highly specific and sensitive tests and the variability between observers is less than that of many diseases and clinical judgments. If a clinical serum test is good enough to replace a tissue biopsy and 
pathologic examination, then it will, no matter how good the biopsy is. There are many historical examples 
in which tissue pathology has been replaced by other kinds of tests and it was not necessary to make such arguments against the use of the biopsy. I would suggest that these sorts of pejorative comments be removed from the introduction and discussion sections. They really don’t add to the argument for using 
biomarkers or serum tests. 
 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 
 Diagnostic Value of Biochemical Markers (NashTest) for the prediction of NASH in Patients with Title: 
Non-Alcoholic Fatty Liver Disease 
 1  9 September 2006 Version: Date: 
 David Kleiner Reviewer: 
Reviewer's report: 
General 
This paper describes the use of a proprietary combination of biochemical assays and demographic/physical 
characteristics to predict whether or not a patient’s liver biopsy will show steatohepatitis. 
------------------------------------------------------------------------------- 
Major Compulsory Revisions (that the author must respond to before a decision on publication can be 
reached) 
1. Methods, p.10, paragraph beginning “Steatohepatitis was defined as a NAS score of 5 or greater.” In this 
study, the diagnosis of NASH is based on the numerical outcome of a recently developed scoring system 
for NASH (ref #14)—in particular the NASH Activity Score (NAS). The authors categorize the fatty liver 
disease based on the value of the NAS, with 0-2 being not diagnostic of NASH, 3-4 being borderline NASH and 5-8 being diagnostic of NASH. This is a misinterpretation of the method. In fact, the data in reference 14 does not support diagnosis by numbers and the authors of that paper note that the NAS is intended for 
use in monitoring changes in liver disease and other clinical situations, and is not intended to replace the 
pathologist’s determination of whether NASH is present or not. The use of the scoring system as proposed 
in ref. 14 is entirely in line with the use of scoring systems in other liver diseases, where scores are used to scale severity and not to determine whether or not the patient has the liver disease under study. To the 
extent that the NAS represents the severity of current liver injury, the proposed NashTest may separate 
those with more severe injury from those with little injury. This would be of great value in clinical trial situations where the investigator might want to enroll those with severe disease first or perhaps for 
identifying patients at greatest risk for progression. However, given that about a third of biopsies in the NAS 
3-4 range in ref. 14 were definite NASH and another third not diagnostic of NASH, the utility of the 
NashTest for identifying patients with bona fide NASH is questionable. The authors need to diagnostically categorize the biopsies according to accepted criteria (which may be found in many publications), independent of the NAS and proceed on that basis or revise the article and test as one which looks at 
severity of injury and does not make claims about diagnosis. 
2. Methods, p.11, paragraph beginning “The new panel, NT…”. There is almost no detail about how the 
base data was combined to give NT. Given that this is intended to be the critical paper that validates the use 
of the assay this is too little detail. The authors have a patent pending on the methodology of deriving the 
NashTest—it is not unreasonable for them to share more of the basic assay and calculation information with 
the readers. The authors need to provide more information on the derivation of this test and show the 
appropriate supporting data. 3. Results, general comment. This comment is related to #2 above. There is no data related to the 
distribution of the NashTest values over the various diagnostic categories or any of the performance 
characteristics of the assay. Leaving aside the issue of whether the cases are grouped appropriately for diagnosis, these are critical data completely missing from this paper. The test is presented as a black box that generates diagnoses. The authors would like to demonstrate their new test as a way to diagnose liver 
disease but offer no data (other than the categorization by the test) on the test itself. Data on the test itself 
needs to be shown. 
------------------------------------------------------------------------------- 
Minor Essential Revisions (such as missing labels on figures, or the wrong use of a term, which the author 
can be trusted to correct) 
4. Methods, p.9 “All patients and controls gave informed consent for the use of data and serum for research 
purposes.” No mention is made of approval of the use of the biopsy for research purposes; this should be 
noted as well along with any institutional oversight of this project. 
5. Results, data presentation. The authors present all of their data in summary tables as means, standard errors and p values. This type of presentation would be enhanced by figures showing the distribution of critical data by category. Box plots are very useful for this purpose and since there are no limitations on 
space for this journal, I would recommend that the authors show as much of the critical data as possible. 
6. Results, Table 1. Fibrosis Stage. The use of F0, F1 etc. to denote fibrosis stage is a peculiarity of the 
Metavir scoring system and should not be used for general designation of fibrosis stage in other systems. 
7. Figure 1C (presumably) is missing. 
------------------------------------------------------------------------------- 
Discretionary Revisions (which the author can choose to ignore) 8. Introduction, p.5 “…there is considerable sampling variability; and high intra- and inter-pathologist 
variability…”. This is a subjective value judgment for which there is no data (despite the references). There 
is no more variability in determination of the diagnosis of NASH than in many other medical diseases that lack highly specific and sensitive tests and the variability between observers is less than that of many diseases and clinical judgments. If a clinical serum test is good enough to replace a tissue biopsy and 
pathologic examination, then it will, no matter how good the biopsy is. There are many historical examples 
in which tissue pathology has been replaced by other kinds of tests and it was not necessary to make such arguments against the use of the biopsy. I would suggest that these sorts of pejorative comments be removed from the introduction and discussion sections. They really don’t add to the argument for using 
biomarkers or serum tests. 
 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