Reviewer's report
Title:Validation of intraluminal and intraperitoneal microdialysis in ischemic small
intestine - Clinical feasibility of equilibration dialysis.
Version: 1Date:24 June 2013
Reviewer: Norbert Cibicek
Reviewer's report:
• Major Compulsory Revisions
1, I disagree with the use of some words. For example „ex vivo“ may be defined
as „out of the living“. The organ must be completely removed from the body and
exist in an artificial medium during the testing procedure. The authors declare
that they used an ex vivo model, but in fact what they did was a determination of
metabolites in microdialysates obtained from ischaemic gut BEFORE it was
removed out of the body (pg. 8). This means we are dealing in fact with sampling
performed in the living system, or „in vivo“. If I just do not understand the
description of the procedure given by the authors (at different places in the
manuscript) and the resecate was placed in the water bath, where the dialysate
was collected, there is no place for „intraperitoneal“ approach – since this must
take place in the peritoneal cavity and not in a sterile cup.
2, Concerning the intraperitoneal position of a catheter, it was defined by
Sommer and Larsen differently (see Fig. 1 in the reference No 9. in the
manuscript, pg. 17). This position requires free placement of the probe in the
peritoneal cavity, not its direct and tight association with the serosa of the gut
with limited access to the peritoneal fluid (what is described in the present
manuscript). Therefore, the latter should be termed „extramural“ position instead.
3, Some statements are false – e.g. the third one in the Background section of
the manuscript. Intestinal luminal application of microdialysis has already been
validated by other authors, at least in the large gut in terms of detection of
ischaemic events.
4, What is the „gut hypothesis“ (pg. 4 in the middle)? Such word connections
need to be clearly defined if they are to be used.
5, Glycerol can by no means be considered a „specific marker of the damage of
the gut epithelia“, as stated in the manuscript. There is a number of different
sources of gut glycerol including dietary triacylglycerols, glycerol enema or
infused lipids. Moreover, various events may contribute to gut glycerol besides
ischaemia. Some of these factors may be found in the latter parts of the
manuscript (pg. 12).
6, If the authors used CMA 600 analyzer, they must have been able to measure
urea as well. Microdialysate urea (added to the perfusion fluid) has recently been
validated for blood perfusion estimation in a number of studies, none of which
have been cited by the authors. This marker would bring important additional
information and enable a more correct interpretation of the results of metabolic
markers. Other validated markers of blood perfusion (such as ethanol) have not
been mentioned in the background.
7, Very importantly, the collection of fractions is wrongly described. For example,
why did the authors collect 10-min fractions and then presented fractions
collected in 30-min intervals? My own experience with gut ischaemia and CMA
600 analyzer tell me that there is a huge problem with the analysis of frozen
samples. After thawing, bubbles are formed in the samples and if there is only 10
microlitres, it is highly probable that the analyzer takes insufficient sample
volume (small dead volume) resulting in very low concentration of the analyte(s).
If the sequence of analyte sis always the same, the last in the row suffers the
most. It is likely that, by default, the last analyte was pyruvate (I experienced this
in my own experiments). If this was the case, the authors may have a plausible
explanation for their rather unexpected and inconsistent results. For instance,
high LP ratio may not be only a result of high lactate, but also very low pyruvate
(the colorimetric principle of the measurement allows the release of very low
results even if there is almost no sample present in the reaction mixture).
Because it would be very difficult (almost impossible) to repeat the analyses from
only 10 microlitres, the authors would have no possiblity to check whether the
sample dilution problem occured. There are more data indicating high probability
of the existence of this problem – very high variability of the results even during
baseline conditions, discrepancy between LP and LG ratios and unexpectably
high baseline values. None of these have been satisfactorily addressed by the
authors in the discussion. By the way, was this problem the reason why the
authors finally decided to put the samples together and analyze them en bloc (in
30-min intervals instead of 10-min intervals)? Or did they publish the means of
three serial values?
8, As to the number of patients and their description, different parts of the
manuscript offer different figures (11, then 21 and finally 20) that are confusing
for a reader. The data (age of the group) must correspond to the group´s
homogeneity, e.g. if there are men and women (two subgroups), two figures
should be provided.
9, The results of sensitivity and specificity cannot be compared when there is no
identical reference. As a reference, 95 % specificity is usually selected. So the
authors should present the values for sensitivity at 95 % specificity, otherwise
“apples are compared with pears”.
10, Levels of glycerol cannot be put as 9 (median) and 34 – 37 (interquartile
range).
11, The statistical analysis is insufficiently described. If the ROC analysis is
performed, there must be a reference (healthy) group / data. These were
obviously the baseline values (however questionable due to very high variability)
– this group must be specified, even though no “sham-operated group” or
“control catheter location” was tested.
12, I can see no reason why the authors selected 68 as the threshold for
intraluminal LP ratio, if the value 109 has better characteristics in terms of
sensitivity and specificity. Is it because it is closer to 20, which was defined in a
number of other studies and confirmed by our group (Cibicek N et al. Physiol
Meas 2010)?
13, The discussion in general is very weak – the authors were unable to
acceptably discuss the metabolic parameters, especially the discrepancies
between the findings of other groups and their data and discrepancies between
different locations (e.g. why the tresholds differ so much between the intraluminal
and “intraperitoneal” markers and why there is so high variability?)
Regarding the equilibration dialysis, the LP and LG ratios (including glycerol)
cannot be equal to 0. It means lactate (and glycerol) must have been equal to 0,
which was not documented in the previous findings (even in the same
manuscript).
14, NJ feeding tube with equilibration dialysis could be considered a feasible
method for clinical purposes provided the results were adequately discussed.
The authors, however, failed to do this.
15, The placement of the probes is not fully delineated. How was the intraluminal
probe implanted? Was there a puncture and/or suture?
16, If there was so great effect of ischaemia on other markers, why glycerol was
unaffected (in the i.p. catheter)?
17, The discussion of LG ratio includes results of glucose obtained by other
groups. However, no information on the perfusion rates (that determine the
recoveries of probes) is provided for the reader, so the given values are of no
use.
The reference No. 22 cannot be used at the beginning of the page 14. The cited
paper did not study the transient increase in glucose concentration - it has only
been speculated by Deeba et al. in an attempt to account for their unexpected
results. The authors cite Deeba et al. here without knowing what they really
meant by additional glucose delivery etc.
• Minor Essential Revisions
18, The article contains numerous english grammatical errors. They are most
often of minor importace (US english vs. British english spelling, spelling in
general, wrong use of articles, missing articles, wrong use of prepositions,
frequently repeated words and/or their incorrect use – e.g. to depict), sometimes,
however, the reader gets confused by long and incomprehensible sentences
(especially the last sentence on the pg. 6). Other sentences and explanations
(such as peritoneal cavity) are redundant or repeated more times in the
paragraphs (such as the absence of a clinical report on the page 4 and 5 or the
major results on the pg. 11). Word connections such as „serosal metabolic
signalling“ on the page 4 are used in a wrong manner – the word „signalling“
means something completely different. The same sentence must have a
reference – how do the authors know that gut motility influences the (recovery of)
metabolic markers? English has to be reviewed by a professional. The authors
most decide if they are to use a „Whipple procedure“ or "Whipple´s procedure“.
Spesificity must be changed for specificity in the figures and tables.
19, There is a number of errors in the References as well. I have identified at
least five (e.g. intraperitioneal, Apperent, reable ...).
20, The key words were not selected properly. ROC analysis should not be
included as a key word, there was no glucose to pyruvate ratio (as stated in the
key words) but lactate to glucose ratio studied by the group.
21, Table 1 contains numbers of patients with different measurements. Why
there were only 12 (13) patients with lactate (pH) measurements and 20 patients
with the measurement of saturation? Is it due to technical reasons (inability to
collect the sample) or was the protocol changed in time?
22, Finally, due to the number of grammatical mistakes, I do not believe that all
(seven!) authors read the manuscript. In such case at least some of them must
have identified the mistakes.
• Discretionary Revisions
None.
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Needs some language corrections before being
published
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare that I have no competing interests.
Reviewer's report
Title:Validation of intraluminal and intraperitoneal microdialysis in ischemic small
intestine - Clinical feasibility of equilibration dialysis.
Version: 1Date:24 June 2013
Reviewer: Norbert Cibicek
Reviewer's report:
• Major Compulsory Revisions
1, I disagree with the use of some words. For example „ex vivo“ may be defined
as „out of the living“. The organ must be completely removed from the body and
exist in an artificial medium during the testing procedure. The authors declare
that they used an ex vivo model, but in fact what they did was a determination of
metabolites in microdialysates obtained from ischaemic gut BEFORE it was
removed out of the body (pg. 8). This means we are dealing in fact with sampling
performed in the living system, or „in vivo“. If I just do not understand the
description of the procedure given by the authors (at different places in the
manuscript) and the resecate was placed in the water bath, where the dialysate
was collected, there is no place for „intraperitoneal“ approach – since this must
take place in the peritoneal cavity and not in a sterile cup.
2, Concerning the intraperitoneal position of a catheter, it was defined by
Sommer and Larsen differently (see Fig. 1 in the reference No 9. in the
manuscript, pg. 17). This position requires free placement of the probe in the
peritoneal cavity, not its direct and tight association with the serosa of the gut
with limited access to the peritoneal fluid (what is described in the present
manuscript). Therefore, the latter should be termed „extramural“ position instead.
3, Some statements are false – e.g. the third one in the Background section of
the manuscript. Intestinal luminal application of microdialysis has already been
validated by other authors, at least in the large gut in terms of detection of
ischaemic events.
4, What is the „gut hypothesis“ (pg. 4 in the middle)? Such word connections
need to be clearly defined if they are to be used.
5, Glycerol can by no means be considered a „specific marker of the damage of
the gut epithelia“, as stated in the manuscript. There is a number of different
sources of gut glycerol including dietary triacylglycerols, glycerol enema or
infused lipids. Moreover, various events may contribute to gut glycerol besides
ischaemia. Some of these factors may be found in the latter parts of the
manuscript (pg. 12).
6, If the authors used CMA 600 analyzer, they must have been able to measure
urea as well. Microdialysate urea (added to the perfusion fluid) has recently been
validated for blood perfusion estimation in a number of studies, none of which
have been cited by the authors. This marker would bring important additional
information and enable a more correct interpretation of the results of metabolic
markers. Other validated markers of blood perfusion (such as ethanol) have not
been mentioned in the background.
7, Very importantly, the collection of fractions is wrongly described. For example,
why did the authors collect 10-min fractions and then presented fractions
collected in 30-min intervals? My own experience with gut ischaemia and CMA
600 analyzer tell me that there is a huge problem with the analysis of frozen
samples. After thawing, bubbles are formed in the samples and if there is only 10
microlitres, it is highly probable that the analyzer takes insufficient sample
volume (small dead volume) resulting in very low concentration of the analyte(s).
If the sequence of analyte sis always the same, the last in the row suffers the
most. It is likely that, by default, the last analyte was pyruvate (I experienced this
in my own experiments). If this was the case, the authors may have a plausible
explanation for their rather unexpected and inconsistent results. For instance,
high LP ratio may not be only a result of high lactate, but also very low pyruvate
(the colorimetric principle of the measurement allows the release of very low
results even if there is almost no sample present in the reaction mixture).
Because it would be very difficult (almost impossible) to repeat the analyses from
only 10 microlitres, the authors would have no possiblity to check whether the
sample dilution problem occured. There are more data indicating high probability
of the existence of this problem – very high variability of the results even during
baseline conditions, discrepancy between LP and LG ratios and unexpectably
high baseline values. None of these have been satisfactorily addressed by the
authors in the discussion. By the way, was this problem the reason why the
authors finally decided to put the samples together and analyze them en bloc (in
30-min intervals instead of 10-min intervals)? Or did they publish the means of
three serial values?
8, As to the number of patients and their description, different parts of the
manuscript offer different figures (11, then 21 and finally 20) that are confusing
for a reader. The data (age of the group) must correspond to the group´s
homogeneity, e.g. if there are men and women (two subgroups), two figures
should be provided.
9, The results of sensitivity and specificity cannot be compared when there is no
identical reference. As a reference, 95 % specificity is usually selected. So the
authors should present the values for sensitivity at 95 % specificity, otherwise
“apples are compared with pears”.
10, Levels of glycerol cannot be put as 9 (median) and 34 – 37 (interquartile
range).
11, The statistical analysis is insufficiently described. If the ROC analysis is
performed, there must be a reference (healthy) group / data. These were
obviously the baseline values (however questionable due to very high variability)
– this group must be specified, even though no “sham-operated group” or
“control catheter location” was tested.
12, I can see no reason why the authors selected 68 as the threshold for
intraluminal LP ratio, if the value 109 has better characteristics in terms of
sensitivity and specificity. Is it because it is closer to 20, which was defined in a
number of other studies and confirmed by our group (Cibicek N et al. Physiol
Meas 2010)?
13, The discussion in general is very weak – the authors were unable to
acceptably discuss the metabolic parameters, especially the discrepancies
between the findings of other groups and their data and discrepancies between
different locations (e.g. why the tresholds differ so much between the intraluminal
and “intraperitoneal” markers and why there is so high variability?)
Regarding the equilibration dialysis, the LP and LG ratios (including glycerol)
cannot be equal to 0. It means lactate (and glycerol) must have been equal to 0,
which was not documented in the previous findings (even in the same
manuscript).
14, NJ feeding tube with equilibration dialysis could be considered a feasible
method for clinical purposes provided the results were adequately discussed.
The authors, however, failed to do this.
15, The placement of the probes is not fully delineated. How was the intraluminal
probe implanted? Was there a puncture and/or suture?
16, If there was so great effect of ischaemia on other markers, why glycerol was
unaffected (in the i.p. catheter)?
17, The discussion of LG ratio includes results of glucose obtained by other
groups. However, no information on the perfusion rates (that determine the
recoveries of probes) is provided for the reader, so the given values are of no
use.
The reference No. 22 cannot be used at the beginning of the page 14. The cited
paper did not study the transient increase in glucose concentration - it has only
been speculated by Deeba et al. in an attempt to account for their unexpected
results. The authors cite Deeba et al. here without knowing what they really
meant by additional glucose delivery etc.
• Minor Essential Revisions
18, The article contains numerous english grammatical errors. They are most
often of minor importace (US english vs. British english spelling, spelling in
general, wrong use of articles, missing articles, wrong use of prepositions,
frequently repeated words and/or their incorrect use – e.g. to depict), sometimes,
however, the reader gets confused by long and incomprehensible sentences
(especially the last sentence on the pg. 6). Other sentences and explanations
(such as peritoneal cavity) are redundant or repeated more times in the
paragraphs (such as the absence of a clinical report on the page 4 and 5 or the
major results on the pg. 11). Word connections such as „serosal metabolic
signalling“ on the page 4 are used in a wrong manner – the word „signalling“
means something completely different. The same sentence must have a
reference – how do the authors know that gut motility influences the (recovery of)
metabolic markers? English has to be reviewed by a professional. The authors
most decide if they are to use a „Whipple procedure“ or "Whipple´s procedure“.
Spesificity must be changed for specificity in the figures and tables.
19, There is a number of errors in the References as well. I have identified at
least five (e.g. intraperitioneal, Apperent, reable ...).
20, The key words were not selected properly. ROC analysis should not be
included as a key word, there was no glucose to pyruvate ratio (as stated in the
key words) but lactate to glucose ratio studied by the group.
21, Table 1 contains numbers of patients with different measurements. Why
there were only 12 (13) patients with lactate (pH) measurements and 20 patients
with the measurement of saturation? Is it due to technical reasons (inability to
collect the sample) or was the protocol changed in time?
22, Finally, due to the number of grammatical mistakes, I do not believe that all
(seven!) authors read the manuscript. In such case at least some of them must
have identified the mistakes.
• Discretionary Revisions
None.
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Needs some language corrections before being
published
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare that I have no competing interests.
Please review this paper.
user
Reviewer's report
Title:Validation of intraluminal and intraperitoneal microdialysis in ischemic small
intestine - Clinical feasibility of equilibration dialysis.
Version: 1Date:24 June 2013
Reviewer: Norbert Cibicek
Reviewer's report:
• Major Compulsory Revisions
1, I disagree with the use of some words. For example „ex vivo“ may be defined
as „out of the living“. The organ must be completely removed from the body and
exist in an artificial medium during the testing procedure. The authors declare
that they used an ex vivo model, but in fact what they did was a determination of
metabolites in microdialysates obtained from ischaemic gut BEFORE it was
removed out of the body (pg. 8). This means we are dealing in fact with sampling
performed in the living system, or „in vivo“. If I just do not understand the
description of the procedure given by the authors (at different places in the
manuscript) and the resecate was placed in the water bath, where the dialysate
was collected, there is no place for „intraperitoneal“ approach – since this must
take place in the peritoneal cavity and not in a sterile cup.
2, Concerning the intraperitoneal position of a catheter, it was defined by
Sommer and Larsen differently (see Fig. 1 in the reference No 9. in the
manuscript, pg. 17). This position requires free placement of the probe in the
peritoneal cavity, not its direct and tight association with the serosa of the gut
with limited access to the peritoneal fluid (what is described in the present
manuscript). Therefore, the latter should be termed „extramural“ position instead.
3, Some statements are false – e.g. the third one in the Background section of
the manuscript. Intestinal luminal application of microdialysis has already been
validated by other authors, at least in the large gut in terms of detection of
ischaemic events.
4, What is the „gut hypothesis“ (pg. 4 in the middle)? Such word connections
need to be clearly defined if they are to be used.
5, Glycerol can by no means be considered a „specific marker of the damage of
the gut epithelia“, as stated in the manuscript. There is a number of different
sources of gut glycerol including dietary triacylglycerols, glycerol enema or
infused lipids. Moreover, various events may contribute to gut glycerol besides
ischaemia. Some of these factors may be found in the latter parts of the
manuscript (pg. 12).
6, If the authors used CMA 600 analyzer, they must have been able to measure
urea as well. Microdialysate urea (added to the perfusion fluid) has recently been
validated for blood perfusion estimation in a number of studies, none of which
have been cited by the authors. This marker would bring important additional
information and enable a more correct interpretation of the results of metabolic
markers. Other validated markers of blood perfusion (such as ethanol) have not
been mentioned in the background.
7, Very importantly, the collection of fractions is wrongly described. For example,
why did the authors collect 10-min fractions and then presented fractions
collected in 30-min intervals? My own experience with gut ischaemia and CMA
600 analyzer tell me that there is a huge problem with the analysis of frozen
samples. After thawing, bubbles are formed in the samples and if there is only 10
microlitres, it is highly probable that the analyzer takes insufficient sample
volume (small dead volume) resulting in very low concentration of the analyte(s).
If the sequence of analyte sis always the same, the last in the row suffers the
most. It is likely that, by default, the last analyte was pyruvate (I experienced this
in my own experiments). If this was the case, the authors may have a plausible
explanation for their rather unexpected and inconsistent results. For instance,
high LP ratio may not be only a result of high lactate, but also very low pyruvate
(the colorimetric principle of the measurement allows the release of very low
results even if there is almost no sample present in the reaction mixture).
Because it would be very difficult (almost impossible) to repeat the analyses from
only 10 microlitres, the authors would have no possiblity to check whether the
sample dilution problem occured. There are more data indicating high probability
of the existence of this problem – very high variability of the results even during
baseline conditions, discrepancy between LP and LG ratios and unexpectably
high baseline values. None of these have been satisfactorily addressed by the
authors in the discussion. By the way, was this problem the reason why the
authors finally decided to put the samples together and analyze them en bloc (in
30-min intervals instead of 10-min intervals)? Or did they publish the means of
three serial values?
8, As to the number of patients and their description, different parts of the
manuscript offer different figures (11, then 21 and finally 20) that are confusing
for a reader. The data (age of the group) must correspond to the group´s
homogeneity, e.g. if there are men and women (two subgroups), two figures
should be provided.
9, The results of sensitivity and specificity cannot be compared when there is no
identical reference. As a reference, 95 % specificity is usually selected. So the
authors should present the values for sensitivity at 95 % specificity, otherwise
“apples are compared with pears”.
10, Levels of glycerol cannot be put as 9 (median) and 34 – 37 (interquartile
range).
11, The statistical analysis is insufficiently described. If the ROC analysis is
performed, there must be a reference (healthy) group / data. These were
obviously the baseline values (however questionable due to very high variability)
– this group must be specified, even though no “sham-operated group” or
“control catheter location” was tested.
12, I can see no reason why the authors selected 68 as the threshold for
intraluminal LP ratio, if the value 109 has better characteristics in terms of
sensitivity and specificity. Is it because it is closer to 20, which was defined in a
number of other studies and confirmed by our group (Cibicek N et al. Physiol
Meas 2010)?
13, The discussion in general is very weak – the authors were unable to
acceptably discuss the metabolic parameters, especially the discrepancies
between the findings of other groups and their data and discrepancies between
different locations (e.g. why the tresholds differ so much between the intraluminal
and “intraperitoneal” markers and why there is so high variability?)
Regarding the equilibration dialysis, the LP and LG ratios (including glycerol)
cannot be equal to 0. It means lactate (and glycerol) must have been equal to 0,
which was not documented in the previous findings (even in the same
manuscript).
14, NJ feeding tube with equilibration dialysis could be considered a feasible
method for clinical purposes provided the results were adequately discussed.
The authors, however, failed to do this.
15, The placement of the probes is not fully delineated. How was the intraluminal
probe implanted? Was there a puncture and/or suture?
16, If there was so great effect of ischaemia on other markers, why glycerol was
unaffected (in the i.p. catheter)?
17, The discussion of LG ratio includes results of glucose obtained by other
groups. However, no information on the perfusion rates (that determine the
recoveries of probes) is provided for the reader, so the given values are of no
use.
The reference No. 22 cannot be used at the beginning of the page 14. The cited
paper did not study the transient increase in glucose concentration - it has only
been speculated by Deeba et al. in an attempt to account for their unexpected
results. The authors cite Deeba et al. here without knowing what they really
meant by additional glucose delivery etc.
• Minor Essential Revisions
18, The article contains numerous english grammatical errors. They are most
often of minor importace (US english vs. British english spelling, spelling in
general, wrong use of articles, missing articles, wrong use of prepositions,
frequently repeated words and/or their incorrect use – e.g. to depict), sometimes,
however, the reader gets confused by long and incomprehensible sentences
(especially the last sentence on the pg. 6). Other sentences and explanations
(such as peritoneal cavity) are redundant or repeated more times in the
paragraphs (such as the absence of a clinical report on the page 4 and 5 or the
major results on the pg. 11). Word connections such as „serosal metabolic
signalling“ on the page 4 are used in a wrong manner – the word „signalling“
means something completely different. The same sentence must have a
reference – how do the authors know that gut motility influences the (recovery of)
metabolic markers? English has to be reviewed by a professional. The authors
most decide if they are to use a „Whipple procedure“ or "Whipple´s procedure“.
Spesificity must be changed for specificity in the figures and tables.
19, There is a number of errors in the References as well. I have identified at
least five (e.g. intraperitioneal, Apperent, reable ...).
20, The key words were not selected properly. ROC analysis should not be
included as a key word, there was no glucose to pyruvate ratio (as stated in the
key words) but lactate to glucose ratio studied by the group.
21, Table 1 contains numbers of patients with different measurements. Why
there were only 12 (13) patients with lactate (pH) measurements and 20 patients
with the measurement of saturation? Is it due to technical reasons (inability to
collect the sample) or was the protocol changed in time?
22, Finally, due to the number of grammatical mistakes, I do not believe that all
(seven!) authors read the manuscript. In such case at least some of them must
have identified the mistakes.
• Discretionary Revisions
None.
Level of interest: An article whose findings are important to those with closely
related research interests
Quality of written English: Needs some language corrections before being
published
Statistical review: No, the manuscript does not need to be seen by a
statistician.
Declaration of competing interests:
I declare that I have no competing interests.
assistant