Reviewer's report
Title:Rectal Biopsies Procedure in Cystic Fibrosis: Technical Aspects and
Patients Perspective for Clinical Trials Feasibility
Version: 1Date:6 November 2012
Reviewer: Frauke Stanke
Reviewer's report:
The manuscript of Servodini, Sousa and colleagues advertises to communicate
insights on rectal biopsies from a technical point of view and from the patient’s
perspective. The authors took care to characterize their biopsy specimens
thoroughly with a variety of techniques including electrochemistry,
immunocytochemistry and histochemistry. However, in contrast to the somewhat
general title that leads the reader to expect a broad spectrum of biopsy
techniques and preparation methods will be compared, reflecting the diverse
procedures in use in the field, the technique is restricted to only one of the two
commonly used biopsy methods of Ussing-chamber based studies on cystic
fibrosis intestinal tissue. The reviewer feels that the manuscript’s contents are of
little value outside of the small community who use forceps biopsies to study the
cystic fibrosis phenotype. Further conclusions by the authors such as the use of
the technique for diagnosing atypical and/or rare cases of cystic fibrosis and its
use as an outcome measure in clinical settings is already well-accepted in the
field.
ICM has been developed for cystic fibrosis diagnosis using the so-called
“Rotterdam protocol”, pioneered by H. de Jonge and colleagues who studied
rectal suction biopsies in open-circuit Ussing chambers in the 1990s (Key
references: Veeze HJ, Sinaasappel M, Bijman J, Bouquet J, De Jonge HR. Ion
transport abnormalities in rectal suction biopsies from children with cystic fibrosis.
Gastroenterology 1991;101:398– 401. Veeze HJ, Halley DJJ, Bijman J, De
Jongste JC, De Jonge HR,
Sinaasappel M. Determinants of mild clinical symptoms in cystic fibrosis patients.
J Clin Invest 1994;93:461– 6.). Alternative protocols have been developed and
implemented in cystic fibrosis diagnosis. A consensus was attempted at the
beginning of the millennium, however, not one but two consensus statements on
ICM have been issued in the Journal of Cystic fibrosis in 2004 (references 13
and 15 of the manuscript).
Currently, these differences are adequately addressed in an editorial by JP
Clancy (Thorax 2010) who writes on ICM: “The procedure has been used in
European CF care and research centres for nearly 20 years, and over this time
some differences have arisen in technique and assay performance that are
site-specific. That being said, the general technique has been shown clearly to
discriminate between CF and non-CF patients and demonstrates a clear
relationship between measured CFTR function and predicted CFTR activity
(based on mechanistic understanding of disease-causing mutations).” In the light
of this comment from 2010, the reviewer feels that the manuscript presented by
Servidoni, Sousa and colleagues in 2012 does not offer new insights on the use
of rectal biopsies in the analysis of cystic fibrosis.
A major limit of the current manuscript that claims to evaluate technical aspects
on rectal biopsies is the limitation to forceps biopsies. The authors have
restricted the data to their method of choice: only forceps biopsies analysed,
suction biopsies are not included in their survey of the procedure or their
tolerance by the patients. As a consequence, this article is of very limited interest
even among those who do perform ICM to diagnose CF. It would be laudable if
the authors could undertake the effort to contact their colleagues in Europe,
using the well-established CF research network, who might be able to provide
access to similar data from centers that use suction biopsy. All centers who do
ICM measure bioelectric properties of their samples, so it should be feasible to
collect at least these in a long overdue European consensus manuscript.
Moreover, recently the question of “sigmoidoscopy vs suction biopsy” has been
addressed by a center who does use both procedures in terms of the patient’s
tolerance, albeit the statement is unfortunately not backed up with systematic
data: Cohen-Cymberknoh M, Yaakov Y, Shoseyov D, Shteyer E, Schachar E,
Rivlin J, Bentur L, Picard E, Aviram M, Israeli E, Kerem E, Wilschanski M.
Evaluation of the intestinal current measurement method as a diagnostic test for
cystic fibrosis. Pediatr Pulmonol. 2012 May 2. doi: 10.1002/ppul.22586. [Epub
ahead of print] report in their article: “Two superficial rectal biopsies
(approximately 4–5 mm in diameter) are obtained by rectal suction biopsy tool
(MMI Instruments, Hackensack, New Jersey) or flexible sigmoidoscopy. The
approach using the former device (biopsy gun) simplifies the process for both the
subject and the investigator team. It is well established that there is no difference
between suction biopsy and forceps biopsy.” (for the latter comment
Cohen-Cymberknoh et al reference Hug MJ, Derichs N, Bronsveld I, Clancy JP.
Measurement of ion transport function in rectal biopsies. In: Amaral MD,
Kunzelmann K, editors. Cystic fibrosis—diagnosis and protocols. Volume I:
approaches to study and correct CFTR defects. New York: Springer Science þ
Business media, LLC; 2011. pp 87– 107).
In the light of the above statements, the reviewer wants to make the following
recommendations to avoid that the extensive work undertaken by Servodini,
Sousa and colleagues will contribute to advance the knowledge in the field:
1. Please make sure that the title, introduction and the aim of the study is clearly
stated and that its limits are recognized at all times (no general biopsy survey,
refinement of an existing technique).
2. Please make sure that more general comments on the benefit of ICM
reference all available options and are not restricted to the authors own work.
Firstly, this is good scientific practice and hence it should go without saying.
Secondly, it will make sure that readers who are not from the field are aware that
there are more choices to be made when setting up ICM than the steps varied by
the authors.
3. As outlined in the point-to-point comments below, the reviewer interprets the
patient’s assessment of the tolerability of the procedure more cautiously based
on the primary data provided by the authors. It might be useful to stratify the
respective part of the results and discussion sections to reflect the ambiguity
adequately. Maybe a physician who has experience in the assessment of coping
strategies and discomfort perception can provide assistance to help objectifying
the author’s interpretation (clinical psychiatrist, child psychiatrist).
Please find below several point-to-point comments:
1. The authors interpret their data on the patient’s perspective with a loving eye:
text reads “most (53%) accept repeating it four more times”. 53% is roughly half
of the sample; “most” would constitute some larger proportion, e.g. three quarters
or more? One could equally conclude from this statement that “many (almost
50%) of patients did not tolerate a repetition of the procedure”. Moreover,
according to the reviewer’s experience, some candidates simply refuse to
undergo the biopsy procedure. Hence, the sample reported upon always suffers
from a selection bias that leads to an underscoring of the fraction of individuals
who do not tolerate the procedure at all.
2. The manuscript shows an unbalanced emphasis on the author’s own preferred
analytic procedure:
Example I: Authors quote Refs 12, 14 (= both their own work) to claim about ICM
” ex vivo assessment …. which we have shown to constitute a sensitive and
robust predictive biomarker for the diagnosis and prognosis of CF disease” (page
4 of manuscript). Refs 12 and 14 are from 2004 and 2012, respectively. There
are more publications, several among them years older than the citations
advertised in the manuscript, most of which use suction biopsies, which all
demonstrate clearly that ICM is a sensitive method. The comment made by JP
Clancy in Thorax 2010 (see above) summarises the state of the art very well.
Example II: Another example is on page 15: text reads “…..Measurements of
CFTR function in rectal biopsy specimens have proven its value in the
fine-diagnosis of patients with milder or "non-classical" forms of CF, in particular
when sweat test results are equivocal or borderline and/or if CFTR-disease
causing mutations are not readily identified by DNA mutation analysis12–14. This
approach also serves as a sensitive test to predict the prognosis when rare
CFTR mutations are not identified by standard screening tests12,14.” Again, the
reviewer urges the authors to reconsider their narrow-minded restriction to their
own work in order to claim the usefulness of their method (see reference 16 of
the manuscript and example II below on the analysis of rare CFTR mutation
genotypes).
Example III: page 15, text reads: “Because functional and biochemical data are
not available for most rare mutations, these specimens can also be used to
establish functional and biochemical correlations with rare CFTR genotypes
(Fig.3B, 3C).” The author’s point is well taken. However, there is functional data
on 17 individuals who are homozygous for deletions, missense, stop or splice
site mutations in the CFTR gene (J Med Genet. 2008 Jan;45(1):47-54.) which is,
to the best knowledge of the reviewer, the most comprehensive study on rare
CFTR mutation genotypes by ICM (publication not quoted by the authors).
3. page 7 - text reads: ”… Data on specimens’ macroscopic evaluation were
obtained for 107 individuals and was averaged for 2-5 biopsies/ individual to
obtain a single value per individual.” The exact properties of the tissue sample
studied in an Ussing chamber determines the outcome of the measurement. If
the authors feel that the endoscopic procedure which allows visual inspection of
the site where the tissue is taken is superior to the blindly performed suction
procedure (as they state on page 16 of their manuscript), a low interindividual
variability of specimens taken from the same patient should prove their point. In
other words: a comparison of the variability of the (bioelectric or other) properties
of a set of samples taken from the same individual might serve as an argument
to choose one method over the other. Can the authors comment on the variability
of their specimens? Can these be used to compare data from centers using the
suction technique?
4. page 9 – text reads “Data were obtained for 75, divided into four age groups:
divided into 4 age groups, namely (yrs): 0-9 (n=21); 10-9 (n=33); 20-29 (n=7);
#30 (n=14). Most patients were children (median age=13 yrs).” This grouping is
fine with respect to the comparison of electrochemical, histochemical and
biochemical data, but unsuitable for the report of the patient’s perspective. It is
unclear who has answered the questions on how the procedure was perceived in
the group of adolescents, who may well have an own opinion (that might be
different from their parent’s statement). It is also unclear from the methods
paragraph whether minors have been asked for their opinion at all or if their
parents were the only ones reporting back to the telephone interview. Besides
these comments, the time span between the biopsy procedure itself and the
interview might have an influence on the assessment of the technique by the
volunteers?
5. page 13 – text reads “Concerning the comparative assessment of the rectal
biopsy procedure with other clinical / diagnosis examinations (Table S5), most of
the individuals classified the overall rectal biopsy procedure as being "more
unpleasant" than sweat test (76%), followed by spirometry (64%) and blood
collection (53.3%),…..” According to the data shown in table S5, in comparison to
ALL techniques offered for comparison by the interviewers, the patients
perceived the rectal biopsy procedure as the more unpleasant one. This is more
noteworthy than currently discussed: none of the diagnostic procedures routinely
employed in cystic fibrosis care are, according to the subjects, worse to endure
than the biopsy procedure. This unequivocal statement of the patients is in
discordance with the assessment of the authors that the procedure is tolerated
very well.
6. page 17 text reads: ”Alternative for bowel preparation in children could be
[…..], which he had previously experienced to be less harmful for the mucosa if
done the day before sigmoidoscopy (data not shown) …..” Who has previously
experienced this alternative treatment? Did the researchers themselves
participate in the study? If yes, the comments of these subjects with respect to
the discomfort evoked by the procedure need to be excluded from the author’s
survey as the researchers will have a positively biased opinion on the technique
(early adapters – phenomenon).
7. page 18 text reads: “in the present study, sedation was used primarily to
reduce anxiety and ensure cooperation”. In line with comment 5, this is also more
noteworthy than currently discussed: “anxiety” and the necessity to “ensure
cooperation” indicates that the technique is not well tolerated.
8. page 26, table 3: there appears to be a strong bias of how many further
biopsies were tolerated depending on who makes the decision (please see also
comment 4 above as the present data display does not disclose who decided
himself / herself and who relied on the decision of the parental guardians). Age
group 0-9 and 10-19: minors and/or their parental guardians decide more
frequent to reject further biopsy-taking than adults (10-29 and >30). Explicitely,
the ratio of [participants who did not agree to have more than one biopsy taken]
to [participants who have agreed to having four biopsies taken] is: [3 to 12], [5 to
15], [0 to 4] and [1 to 9] in the four age groups, translating as a rejection rate of [1
out of 5], [1 out of 4], [none], [1 out of 10]. This strongly implies that parental
guardians perceived the technique as sufficiently discomfortable to reject further
participation at this point. This is not addressed in the present manuscript,
however, it might well be relevant when a research setting (and not a diagnostic
procedure) is attempted. Were adult non-CF volunteers recruited from the
scientific staff? See comment 6 above.
Level of interest: An article of limited interest
Quality of written English: Acceptable
Statistical review: Yes, and I have assessed the statistics in my report.
Declaration of competing interests:
I declare that I have no competing interests.
Reviewer's report
Title:Rectal Biopsies Procedure in Cystic Fibrosis: Technical Aspects and
Patients Perspective for Clinical Trials Feasibility
Version: 1Date:6 November 2012
Reviewer: Frauke Stanke
Reviewer's report:
The manuscript of Servodini, Sousa and colleagues advertises to communicate
insights on rectal biopsies from a technical point of view and from the patient’s
perspective. The authors took care to characterize their biopsy specimens
thoroughly with a variety of techniques including electrochemistry,
immunocytochemistry and histochemistry. However, in contrast to the somewhat
general title that leads the reader to expect a broad spectrum of biopsy
techniques and preparation methods will be compared, reflecting the diverse
procedures in use in the field, the technique is restricted to only one of the two
commonly used biopsy methods of Ussing-chamber based studies on cystic
fibrosis intestinal tissue. The reviewer feels that the manuscript’s contents are of
little value outside of the small community who use forceps biopsies to study the
cystic fibrosis phenotype. Further conclusions by the authors such as the use of
the technique for diagnosing atypical and/or rare cases of cystic fibrosis and its
use as an outcome measure in clinical settings is already well-accepted in the
field.
ICM has been developed for cystic fibrosis diagnosis using the so-called
“Rotterdam protocol”, pioneered by H. de Jonge and colleagues who studied
rectal suction biopsies in open-circuit Ussing chambers in the 1990s (Key
references: Veeze HJ, Sinaasappel M, Bijman J, Bouquet J, De Jonge HR. Ion
transport abnormalities in rectal suction biopsies from children with cystic fibrosis.
Gastroenterology 1991;101:398– 401. Veeze HJ, Halley DJJ, Bijman J, De
Jongste JC, De Jonge HR,
Sinaasappel M. Determinants of mild clinical symptoms in cystic fibrosis patients.
J Clin Invest 1994;93:461– 6.). Alternative protocols have been developed and
implemented in cystic fibrosis diagnosis. A consensus was attempted at the
beginning of the millennium, however, not one but two consensus statements on
ICM have been issued in the Journal of Cystic fibrosis in 2004 (references 13
and 15 of the manuscript).
Currently, these differences are adequately addressed in an editorial by JP
Clancy (Thorax 2010) who writes on ICM: “The procedure has been used in
European CF care and research centres for nearly 20 years, and over this time
some differences have arisen in technique and assay performance that are
site-specific. That being said, the general technique has been shown clearly to
discriminate between CF and non-CF patients and demonstrates a clear
relationship between measured CFTR function and predicted CFTR activity
(based on mechanistic understanding of disease-causing mutations).” In the light
of this comment from 2010, the reviewer feels that the manuscript presented by
Servidoni, Sousa and colleagues in 2012 does not offer new insights on the use
of rectal biopsies in the analysis of cystic fibrosis.
A major limit of the current manuscript that claims to evaluate technical aspects
on rectal biopsies is the limitation to forceps biopsies. The authors have
restricted the data to their method of choice: only forceps biopsies analysed,
suction biopsies are not included in their survey of the procedure or their
tolerance by the patients. As a consequence, this article is of very limited interest
even among those who do perform ICM to diagnose CF. It would be laudable if
the authors could undertake the effort to contact their colleagues in Europe,
using the well-established CF research network, who might be able to provide
access to similar data from centers that use suction biopsy. All centers who do
ICM measure bioelectric properties of their samples, so it should be feasible to
collect at least these in a long overdue European consensus manuscript.
Moreover, recently the question of “sigmoidoscopy vs suction biopsy” has been
addressed by a center who does use both procedures in terms of the patient’s
tolerance, albeit the statement is unfortunately not backed up with systematic
data: Cohen-Cymberknoh M, Yaakov Y, Shoseyov D, Shteyer E, Schachar E,
Rivlin J, Bentur L, Picard E, Aviram M, Israeli E, Kerem E, Wilschanski M.
Evaluation of the intestinal current measurement method as a diagnostic test for
cystic fibrosis. Pediatr Pulmonol. 2012 May 2. doi: 10.1002/ppul.22586. [Epub
ahead of print] report in their article: “Two superficial rectal biopsies
(approximately 4–5 mm in diameter) are obtained by rectal suction biopsy tool
(MMI Instruments, Hackensack, New Jersey) or flexible sigmoidoscopy. The
approach using the former device (biopsy gun) simplifies the process for both the
subject and the investigator team. It is well established that there is no difference
between suction biopsy and forceps biopsy.” (for the latter comment
Cohen-Cymberknoh et al reference Hug MJ, Derichs N, Bronsveld I, Clancy JP.
Measurement of ion transport function in rectal biopsies. In: Amaral MD,
Kunzelmann K, editors. Cystic fibrosis—diagnosis and protocols. Volume I:
approaches to study and correct CFTR defects. New York: Springer Science þ
Business media, LLC; 2011. pp 87– 107).
In the light of the above statements, the reviewer wants to make the following
recommendations to avoid that the extensive work undertaken by Servodini,
Sousa and colleagues will contribute to advance the knowledge in the field:
1. Please make sure that the title, introduction and the aim of the study is clearly
stated and that its limits are recognized at all times (no general biopsy survey,
refinement of an existing technique).
2. Please make sure that more general comments on the benefit of ICM
reference all available options and are not restricted to the authors own work.
Firstly, this is good scientific practice and hence it should go without saying.
Secondly, it will make sure that readers who are not from the field are aware that
there are more choices to be made when setting up ICM than the steps varied by
the authors.
3. As outlined in the point-to-point comments below, the reviewer interprets the
patient’s assessment of the tolerability of the procedure more cautiously based
on the primary data provided by the authors. It might be useful to stratify the
respective part of the results and discussion sections to reflect the ambiguity
adequately. Maybe a physician who has experience in the assessment of coping
strategies and discomfort perception can provide assistance to help objectifying
the author’s interpretation (clinical psychiatrist, child psychiatrist).
Please find below several point-to-point comments:
1. The authors interpret their data on the patient’s perspective with a loving eye:
text reads “most (53%) accept repeating it four more times”. 53% is roughly half
of the sample; “most” would constitute some larger proportion, e.g. three quarters
or more? One could equally conclude from this statement that “many (almost
50%) of patients did not tolerate a repetition of the procedure”. Moreover,
according to the reviewer’s experience, some candidates simply refuse to
undergo the biopsy procedure. Hence, the sample reported upon always suffers
from a selection bias that leads to an underscoring of the fraction of individuals
who do not tolerate the procedure at all.
2. The manuscript shows an unbalanced emphasis on the author’s own preferred
analytic procedure:
Example I: Authors quote Refs 12, 14 (= both their own work) to claim about ICM
” ex vivo assessment …. which we have shown to constitute a sensitive and
robust predictive biomarker for the diagnosis and prognosis of CF disease” (page
4 of manuscript). Refs 12 and 14 are from 2004 and 2012, respectively. There
are more publications, several among them years older than the citations
advertised in the manuscript, most of which use suction biopsies, which all
demonstrate clearly that ICM is a sensitive method. The comment made by JP
Clancy in Thorax 2010 (see above) summarises the state of the art very well.
Example II: Another example is on page 15: text reads “…..Measurements of
CFTR function in rectal biopsy specimens have proven its value in the
fine-diagnosis of patients with milder or "non-classical" forms of CF, in particular
when sweat test results are equivocal or borderline and/or if CFTR-disease
causing mutations are not readily identified by DNA mutation analysis12–14. This
approach also serves as a sensitive test to predict the prognosis when rare
CFTR mutations are not identified by standard screening tests12,14.” Again, the
reviewer urges the authors to reconsider their narrow-minded restriction to their
own work in order to claim the usefulness of their method (see reference 16 of
the manuscript and example II below on the analysis of rare CFTR mutation
genotypes).
Example III: page 15, text reads: “Because functional and biochemical data are
not available for most rare mutations, these specimens can also be used to
establish functional and biochemical correlations with rare CFTR genotypes
(Fig.3B, 3C).” The author’s point is well taken. However, there is functional data
on 17 individuals who are homozygous for deletions, missense, stop or splice
site mutations in the CFTR gene (J Med Genet. 2008 Jan;45(1):47-54.) which is,
to the best knowledge of the reviewer, the most comprehensive study on rare
CFTR mutation genotypes by ICM (publication not quoted by the authors).
3. page 7 - text reads: ”… Data on specimens’ macroscopic evaluation were
obtained for 107 individuals and was averaged for 2-5 biopsies/ individual to
obtain a single value per individual.” The exact properties of the tissue sample
studied in an Ussing chamber determines the outcome of the measurement. If
the authors feel that the endoscopic procedure which allows visual inspection of
the site where the tissue is taken is superior to the blindly performed suction
procedure (as they state on page 16 of their manuscript), a low interindividual
variability of specimens taken from the same patient should prove their point. In
other words: a comparison of the variability of the (bioelectric or other) properties
of a set of samples taken from the same individual might serve as an argument
to choose one method over the other. Can the authors comment on the variability
of their specimens? Can these be used to compare data from centers using the
suction technique?
4. page 9 – text reads “Data were obtained for 75, divided into four age groups:
divided into 4 age groups, namely (yrs): 0-9 (n=21); 10-9 (n=33); 20-29 (n=7);
#30 (n=14). Most patients were children (median age=13 yrs).” This grouping is
fine with respect to the comparison of electrochemical, histochemical and
biochemical data, but unsuitable for the report of the patient’s perspective. It is
unclear who has answered the questions on how the procedure was perceived in
the group of adolescents, who may well have an own opinion (that might be
different from their parent’s statement). It is also unclear from the methods
paragraph whether minors have been asked for their opinion at all or if their
parents were the only ones reporting back to the telephone interview. Besides
these comments, the time span between the biopsy procedure itself and the
interview might have an influence on the assessment of the technique by the
volunteers?
5. page 13 – text reads “Concerning the comparative assessment of the rectal
biopsy procedure with other clinical / diagnosis examinations (Table S5), most of
the individuals classified the overall rectal biopsy procedure as being "more
unpleasant" than sweat test (76%), followed by spirometry (64%) and blood
collection (53.3%),…..” According to the data shown in table S5, in comparison to
ALL techniques offered for comparison by the interviewers, the patients
perceived the rectal biopsy procedure as the more unpleasant one. This is more
noteworthy than currently discussed: none of the diagnostic procedures routinely
employed in cystic fibrosis care are, according to the subjects, worse to endure
than the biopsy procedure. This unequivocal statement of the patients is in
discordance with the assessment of the authors that the procedure is tolerated
very well.
6. page 17 text reads: ”Alternative for bowel preparation in children could be
[…..], which he had previously experienced to be less harmful for the mucosa if
done the day before sigmoidoscopy (data not shown) …..” Who has previously
experienced this alternative treatment? Did the researchers themselves
participate in the study? If yes, the comments of these subjects with respect to
the discomfort evoked by the procedure need to be excluded from the author’s
survey as the researchers will have a positively biased opinion on the technique
(early adapters – phenomenon).
7. page 18 text reads: “in the present study, sedation was used primarily to
reduce anxiety and ensure cooperation”. In line with comment 5, this is also more
noteworthy than currently discussed: “anxiety” and the necessity to “ensure
cooperation” indicates that the technique is not well tolerated.
8. page 26, table 3: there appears to be a strong bias of how many further
biopsies were tolerated depending on who makes the decision (please see also
comment 4 above as the present data display does not disclose who decided
himself / herself and who relied on the decision of the parental guardians). Age
group 0-9 and 10-19: minors and/or their parental guardians decide more
frequent to reject further biopsy-taking than adults (10-29 and >30). Explicitely,
the ratio of [participants who did not agree to have more than one biopsy taken]
to [participants who have agreed to having four biopsies taken] is: [3 to 12], [5 to
15], [0 to 4] and [1 to 9] in the four age groups, translating as a rejection rate of [1
out of 5], [1 out of 4], [none], [1 out of 10]. This strongly implies that parental
guardians perceived the technique as sufficiently discomfortable to reject further
participation at this point. This is not addressed in the present manuscript,
however, it might well be relevant when a research setting (and not a diagnostic
procedure) is attempted. Were adult non-CF volunteers recruited from the
scientific staff? See comment 6 above.
Level of interest: An article of limited interest
Quality of written English: Acceptable
Statistical review: Yes, and I have assessed the statistics in my report.
Declaration of competing interests:
I declare that I have no competing interests.
Please review this paper.
user
Reviewer's report
Title:Rectal Biopsies Procedure in Cystic Fibrosis: Technical Aspects and
Patients Perspective for Clinical Trials Feasibility
Version: 1Date:6 November 2012
Reviewer: Frauke Stanke
Reviewer's report:
The manuscript of Servodini, Sousa and colleagues advertises to communicate
insights on rectal biopsies from a technical point of view and from the patient’s
perspective. The authors took care to characterize their biopsy specimens
thoroughly with a variety of techniques including electrochemistry,
immunocytochemistry and histochemistry. However, in contrast to the somewhat
general title that leads the reader to expect a broad spectrum of biopsy
techniques and preparation methods will be compared, reflecting the diverse
procedures in use in the field, the technique is restricted to only one of the two
commonly used biopsy methods of Ussing-chamber based studies on cystic
fibrosis intestinal tissue. The reviewer feels that the manuscript’s contents are of
little value outside of the small community who use forceps biopsies to study the
cystic fibrosis phenotype. Further conclusions by the authors such as the use of
the technique for diagnosing atypical and/or rare cases of cystic fibrosis and its
use as an outcome measure in clinical settings is already well-accepted in the
field.
ICM has been developed for cystic fibrosis diagnosis using the so-called
“Rotterdam protocol”, pioneered by H. de Jonge and colleagues who studied
rectal suction biopsies in open-circuit Ussing chambers in the 1990s (Key
references: Veeze HJ, Sinaasappel M, Bijman J, Bouquet J, De Jonge HR. Ion
transport abnormalities in rectal suction biopsies from children with cystic fibrosis.
Gastroenterology 1991;101:398– 401. Veeze HJ, Halley DJJ, Bijman J, De
Jongste JC, De Jonge HR,
Sinaasappel M. Determinants of mild clinical symptoms in cystic fibrosis patients.
J Clin Invest 1994;93:461– 6.). Alternative protocols have been developed and
implemented in cystic fibrosis diagnosis. A consensus was attempted at the
beginning of the millennium, however, not one but two consensus statements on
ICM have been issued in the Journal of Cystic fibrosis in 2004 (references 13
and 15 of the manuscript).
Currently, these differences are adequately addressed in an editorial by JP
Clancy (Thorax 2010) who writes on ICM: “The procedure has been used in
European CF care and research centres for nearly 20 years, and over this time
some differences have arisen in technique and assay performance that are
site-specific. That being said, the general technique has been shown clearly to
discriminate between CF and non-CF patients and demonstrates a clear
relationship between measured CFTR function and predicted CFTR activity
(based on mechanistic understanding of disease-causing mutations).” In the light
of this comment from 2010, the reviewer feels that the manuscript presented by
Servidoni, Sousa and colleagues in 2012 does not offer new insights on the use
of rectal biopsies in the analysis of cystic fibrosis.
A major limit of the current manuscript that claims to evaluate technical aspects
on rectal biopsies is the limitation to forceps biopsies. The authors have
restricted the data to their method of choice: only forceps biopsies analysed,
suction biopsies are not included in their survey of the procedure or their
tolerance by the patients. As a consequence, this article is of very limited interest
even among those who do perform ICM to diagnose CF. It would be laudable if
the authors could undertake the effort to contact their colleagues in Europe,
using the well-established CF research network, who might be able to provide
access to similar data from centers that use suction biopsy. All centers who do
ICM measure bioelectric properties of their samples, so it should be feasible to
collect at least these in a long overdue European consensus manuscript.
Moreover, recently the question of “sigmoidoscopy vs suction biopsy” has been
addressed by a center who does use both procedures in terms of the patient’s
tolerance, albeit the statement is unfortunately not backed up with systematic
data: Cohen-Cymberknoh M, Yaakov Y, Shoseyov D, Shteyer E, Schachar E,
Rivlin J, Bentur L, Picard E, Aviram M, Israeli E, Kerem E, Wilschanski M.
Evaluation of the intestinal current measurement method as a diagnostic test for
cystic fibrosis. Pediatr Pulmonol. 2012 May 2. doi: 10.1002/ppul.22586. [Epub
ahead of print] report in their article: “Two superficial rectal biopsies
(approximately 4–5 mm in diameter) are obtained by rectal suction biopsy tool
(MMI Instruments, Hackensack, New Jersey) or flexible sigmoidoscopy. The
approach using the former device (biopsy gun) simplifies the process for both the
subject and the investigator team. It is well established that there is no difference
between suction biopsy and forceps biopsy.” (for the latter comment
Cohen-Cymberknoh et al reference Hug MJ, Derichs N, Bronsveld I, Clancy JP.
Measurement of ion transport function in rectal biopsies. In: Amaral MD,
Kunzelmann K, editors. Cystic fibrosis—diagnosis and protocols. Volume I:
approaches to study and correct CFTR defects. New York: Springer Science þ
Business media, LLC; 2011. pp 87– 107).
In the light of the above statements, the reviewer wants to make the following
recommendations to avoid that the extensive work undertaken by Servodini,
Sousa and colleagues will contribute to advance the knowledge in the field:
1. Please make sure that the title, introduction and the aim of the study is clearly
stated and that its limits are recognized at all times (no general biopsy survey,
refinement of an existing technique).
2. Please make sure that more general comments on the benefit of ICM
reference all available options and are not restricted to the authors own work.
Firstly, this is good scientific practice and hence it should go without saying.
Secondly, it will make sure that readers who are not from the field are aware that
there are more choices to be made when setting up ICM than the steps varied by
the authors.
3. As outlined in the point-to-point comments below, the reviewer interprets the
patient’s assessment of the tolerability of the procedure more cautiously based
on the primary data provided by the authors. It might be useful to stratify the
respective part of the results and discussion sections to reflect the ambiguity
adequately. Maybe a physician who has experience in the assessment of coping
strategies and discomfort perception can provide assistance to help objectifying
the author’s interpretation (clinical psychiatrist, child psychiatrist).
Please find below several point-to-point comments:
1. The authors interpret their data on the patient’s perspective with a loving eye:
text reads “most (53%) accept repeating it four more times”. 53% is roughly half
of the sample; “most” would constitute some larger proportion, e.g. three quarters
or more? One could equally conclude from this statement that “many (almost
50%) of patients did not tolerate a repetition of the procedure”. Moreover,
according to the reviewer’s experience, some candidates simply refuse to
undergo the biopsy procedure. Hence, the sample reported upon always suffers
from a selection bias that leads to an underscoring of the fraction of individuals
who do not tolerate the procedure at all.
2. The manuscript shows an unbalanced emphasis on the author’s own preferred
analytic procedure:
Example I: Authors quote Refs 12, 14 (= both their own work) to claim about ICM
” ex vivo assessment …. which we have shown to constitute a sensitive and
robust predictive biomarker for the diagnosis and prognosis of CF disease” (page
4 of manuscript). Refs 12 and 14 are from 2004 and 2012, respectively. There
are more publications, several among them years older than the citations
advertised in the manuscript, most of which use suction biopsies, which all
demonstrate clearly that ICM is a sensitive method. The comment made by JP
Clancy in Thorax 2010 (see above) summarises the state of the art very well.
Example II: Another example is on page 15: text reads “…..Measurements of
CFTR function in rectal biopsy specimens have proven its value in the
fine-diagnosis of patients with milder or "non-classical" forms of CF, in particular
when sweat test results are equivocal or borderline and/or if CFTR-disease
causing mutations are not readily identified by DNA mutation analysis12–14. This
approach also serves as a sensitive test to predict the prognosis when rare
CFTR mutations are not identified by standard screening tests12,14.” Again, the
reviewer urges the authors to reconsider their narrow-minded restriction to their
own work in order to claim the usefulness of their method (see reference 16 of
the manuscript and example II below on the analysis of rare CFTR mutation
genotypes).
Example III: page 15, text reads: “Because functional and biochemical data are
not available for most rare mutations, these specimens can also be used to
establish functional and biochemical correlations with rare CFTR genotypes
(Fig.3B, 3C).” The author’s point is well taken. However, there is functional data
on 17 individuals who are homozygous for deletions, missense, stop or splice
site mutations in the CFTR gene (J Med Genet. 2008 Jan;45(1):47-54.) which is,
to the best knowledge of the reviewer, the most comprehensive study on rare
CFTR mutation genotypes by ICM (publication not quoted by the authors).
3. page 7 - text reads: ”… Data on specimens’ macroscopic evaluation were
obtained for 107 individuals and was averaged for 2-5 biopsies/ individual to
obtain a single value per individual.” The exact properties of the tissue sample
studied in an Ussing chamber determines the outcome of the measurement. If
the authors feel that the endoscopic procedure which allows visual inspection of
the site where the tissue is taken is superior to the blindly performed suction
procedure (as they state on page 16 of their manuscript), a low interindividual
variability of specimens taken from the same patient should prove their point. In
other words: a comparison of the variability of the (bioelectric or other) properties
of a set of samples taken from the same individual might serve as an argument
to choose one method over the other. Can the authors comment on the variability
of their specimens? Can these be used to compare data from centers using the
suction technique?
4. page 9 – text reads “Data were obtained for 75, divided into four age groups:
divided into 4 age groups, namely (yrs): 0-9 (n=21); 10-9 (n=33); 20-29 (n=7);
#30 (n=14). Most patients were children (median age=13 yrs).” This grouping is
fine with respect to the comparison of electrochemical, histochemical and
biochemical data, but unsuitable for the report of the patient’s perspective. It is
unclear who has answered the questions on how the procedure was perceived in
the group of adolescents, who may well have an own opinion (that might be
different from their parent’s statement). It is also unclear from the methods
paragraph whether minors have been asked for their opinion at all or if their
parents were the only ones reporting back to the telephone interview. Besides
these comments, the time span between the biopsy procedure itself and the
interview might have an influence on the assessment of the technique by the
volunteers?
5. page 13 – text reads “Concerning the comparative assessment of the rectal
biopsy procedure with other clinical / diagnosis examinations (Table S5), most of
the individuals classified the overall rectal biopsy procedure as being "more
unpleasant" than sweat test (76%), followed by spirometry (64%) and blood
collection (53.3%),…..” According to the data shown in table S5, in comparison to
ALL techniques offered for comparison by the interviewers, the patients
perceived the rectal biopsy procedure as the more unpleasant one. This is more
noteworthy than currently discussed: none of the diagnostic procedures routinely
employed in cystic fibrosis care are, according to the subjects, worse to endure
than the biopsy procedure. This unequivocal statement of the patients is in
discordance with the assessment of the authors that the procedure is tolerated
very well.
6. page 17 text reads: ”Alternative for bowel preparation in children could be
[…..], which he had previously experienced to be less harmful for the mucosa if
done the day before sigmoidoscopy (data not shown) …..” Who has previously
experienced this alternative treatment? Did the researchers themselves
participate in the study? If yes, the comments of these subjects with respect to
the discomfort evoked by the procedure need to be excluded from the author’s
survey as the researchers will have a positively biased opinion on the technique
(early adapters – phenomenon).
7. page 18 text reads: “in the present study, sedation was used primarily to
reduce anxiety and ensure cooperation”. In line with comment 5, this is also more
noteworthy than currently discussed: “anxiety” and the necessity to “ensure
cooperation” indicates that the technique is not well tolerated.
8. page 26, table 3: there appears to be a strong bias of how many further
biopsies were tolerated depending on who makes the decision (please see also
comment 4 above as the present data display does not disclose who decided
himself / herself and who relied on the decision of the parental guardians). Age
group 0-9 and 10-19: minors and/or their parental guardians decide more
frequent to reject further biopsy-taking than adults (10-29 and >30). Explicitely,
the ratio of [participants who did not agree to have more than one biopsy taken]
to [participants who have agreed to having four biopsies taken] is: [3 to 12], [5 to
15], [0 to 4] and [1 to 9] in the four age groups, translating as a rejection rate of [1
out of 5], [1 out of 4], [none], [1 out of 10]. This strongly implies that parental
guardians perceived the technique as sufficiently discomfortable to reject further
participation at this point. This is not addressed in the present manuscript,
however, it might well be relevant when a research setting (and not a diagnostic
procedure) is attempted. Were adult non-CF volunteers recruited from the
scientific staff? See comment 6 above.
Level of interest: An article of limited interest
Quality of written English: Acceptable
Statistical review: Yes, and I have assessed the statistics in my report.
Declaration of competing interests:
I declare that I have no competing interests.
assistant