Review for 10.1186/1471-230X-7-32 (2007)

Reviewer's report 
 Grey scale enhancement by a new self-made contrast agent in early cirrhotic stage of rabbit liver Title: 
 3  11 April 2007 Version: Date: 
 John H Kalbfleisch Reviewer: 
Reviewer's report: 
General 
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Major Compulsory Revisions (that the author must respond to before a decision on publication can be reached) 
Review Conslusion : I find the manuscript interesting and would reluctantly favor publication. However, 
statistical analysis remains problematic. 
Apparently, statistical analysis does not change in the revised manuscript. There are 12 animals and five 
disease stages (F0 through F4). Each animal was measured at each stage unless the animal could not 
contribute. A key statistical procedure in the methods section is vague (ANOVA). The ANOVA, dictated by the design, must have a stage factor and an animal factor. Further, because some animals contribute to 
only a few stages, the ANOVA using all data values (n=47) would be performed by GLM with both stage and 
animal factors. Below I illustrate statistical analysis appropriate for the study design for HVAT and 
HA-HVTT, and show how these results compare with results in the manuscript – there are differences. 
Revised manuscript figures 3(a) and 3(b) were used to approximate 47 individual data values. These values 
were summarized by stage and the mean values in the reviewer analysis are in good agreement with 
manuscript stage mean levels (tables below). The data was analyzed with GLM-ANOVA, the 
Tukey-pairwise-comparison procedure and the paired-t test (since some investigators would use this 
method). Additionally, the 30 measurements of the 6 animals with complete stage data were analyzed in 
the same fashion. As shown in the tables, the reviewer analysis results do not entirely agree with result statements in the manuscript. For example, for both HVAT and HA-HVTT the authors do not find statistical differences among F0 to F3 (second paragraph in results). Interestingly, ANOVA-GLM and 
Tukey-comparisons show F0 is statistically different from F1, F2 and F3; using either the set of 47 values or 
the subset of 30 values. The two new figures (3a and 3b) show that all 12 animals decrease from F0 to F1; 
this agrees with the reviewer analysis but is in conflict with stated manuscript results. Interestingly, the reviewer results tend to further support the research hypothesis of the study. 
Table 1 HVAT data analysis summary. 
Author Analyses by Reviewer 
47 values GLM ANOVA 47 values GLM ANOVA 30 values 
---------------------------------------------------------------- Stage N Mean Mean LS Mean Mean F0 12 12.542 12.515 12.515 12.342 
F1 12 11.587 11.588 11.585 11.425 
F2 9 11.331 11.330 11.319 11.200 F3 8 9.896 9.889 9.865 9.960 F4 6 8.743 8.773 8.863 8.773 
T-test paired-T Tukey paired-T Tukey 
result “p” “p” “p” “p” F0 vs F1 NS 0.001 0.0051 0.001 0.1201 
F0 vs F2 NS 0.001 0.0011 0.010 0.0346 
F0 vs F3 NS 0.001 0.0001 0.001 0.0001 F0 vs F4 0.001 0.001 0.0001 0.001 0.0001 F1 vs F2 NS 0.280 0.8645 0.525 0.9693 
F1 vs F3 NS 0.001 0.0001 0.003 0.0049 
F1 vs F4 0.001 0.003 0.0001 0.003 0.0001 
F2 vs F3 NS 0.002 0.0002 0.005 0.0193 
F2 vs F4 0.001 0.002 0.0001 0.002 0.0001 
F3 vs F4 0.044 0.026 0.0346 0.026 0.0266 
---------------------------------------------------------------- Note: Analysis by reviewer is based on values estimated from manuscript figure 3(a). GLM = general linear model, LS = least 
Squares, T-test = procedure indicated in manuscript. 
Table 2 HA-NHTT data analysis summary. 
Author Analyses by Reviewer 
47 values GLM ANOVA 47 values GLM ANOVA 30 values 
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Stage N Mean Mean LS Mean Mean 
F0 12 8.677 8.660 8.660 8.522 F1 12 7.308 7.305 7.305 7.115 F2 9 6.901 6.897 6.823 6.912 
F3 8 6.495 6.513 6.400 6.900 
F4 6 5.165 5.213 5.148 5.213 
T-test paired-T Tukey paired-T Tukey 
result “p” “p” “p” “p” 
F0 vs F1 NS 0.001 0.0030 0.001 0.0352 F0 vs F2 NS 0.001 0.0003 0.002 0.0131 F0 vs F3 NS 0.005 0.0001 0.009 0.0124 
F0 vs F4 <0.001 0.002 0.0001 0.002 0.0001 
F1 vs F2 NS 0.179 0.7052 0.638 0.9904 F1 vs F3 NS 0.176 0.1723 0.655 0.9880 F1 vs F4 0.022 0.035 0.0002 0.035 0.0030 
F2 vs F3 NS 0.260 0.8342 0.968 0.9999 
F2 vs F4 0.004 0.020 0.0062 0.020 0.0085 F3 vs F4 0.003 0.018 0.0678 0.018 0.0090 ---------------------------------------------------------------- 
Note: Analysis by reviewer is based on values estimated from 
manuscript figure 3(b). GLM = general linear model, LS = least Squares, T-test = procedure indicated in manuscript. 
Reviewer Explanation 
The comparison of means between any two stages should be performed on a paired-design basis because 
animals contribute data to both stages. The T-test, which is the analysis used by the authors, compares two 
group means for a design where animals in the comparison groups are different. The paired-T test 
appropriately compares means when the same animals contribute a value to each comparison group. Conclusions of the manuscript T-test and the reviewer paired-T test are different. If the initial analysis is 
ANOVA, the study design dictates accounting for “stage” and “animal.” Since only 6 of 12 animals are 
represented in every stage, the initial analysis of variance was performed by GLM and subsequent pairwise 
comparisons were assessed with Tukey’s procedure. The GLM-ANOVA / Tukey results (see tables) also 
differ from current manuscript results. Further, a 1-way ANOVA would assume there are 47 different animals – certainly not the case in this study design. The reviewer analysis is in conflict with statistical 
analysis advice given to the authors. This discrepancy could result if the design was not clearly identified to 
the statistical consultant. 
My assessment still advocates all of the responses be analyzed with statistical procedures accounting for 
the fact that animals contribute values to several stages (a paired-design in fashion). I cannot comment on 
analysis of PSI measures in the last paragraph of the results section (individual data is absent), but a re-analysis could be in order. 
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Minor Essential Revisions (such as missing labels on figures, or the wrong use of a term, which the author can be trusted to correct) 
PSI measures are shown in Fig.5, not 3a or 3b (last paragraph in results). 
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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 whose findings are important to those with closely related research interests Level of interest: 
 Acceptable Quality of written English: 
 Yes, and I have assessed the statistics in my report. Statistical review: 
Declaration of competing interests: 
I declare that I have no competing interests.