Recently, Sousa and colleagues [1] published in The Journal their data on 100 cystic fibrosis patients analysed by intestinal current measurement to conclude that “Determination of CFTR-mediated Cl- secretion in rectal biopsies is demonstrated here to be a sensitive, reproducible and robust predictive biomarker for the diagnosis and prognosis of CF. The method also has very high potential for (pre-)clinical trials of CFTR-modulator therapies.” I fully agree with the author’s assessment, a deduction that somewhat mirrors the conclusion from an earlier ICM study on 102 CF patients of Derichs and colleagues [2] who have noticed that their work “ […] underlines the diagnostic value of ICM […..]. ICM is an important tool for functional assessment in CFTR mutations of unknown clinical relevance.” Sousa et al [1] and Derichs et al [2] have studied a similar sample size, indicating that both studies have a similar power to detect the value of ICM for CF diagnosis and further usefulness of the technique for functional assessment of CFTR dysfunction. This makes it easy to understand that both authors welcome the use of ICM in CF research. However, these two studies use different protocols for getting the samples that are analysed by ICM (forceps biopsies in [1] versus suction biopsy in [2]) and differ in the experimental set-up for the intestinal current measurement (open circuit [1] vs short circuit [2] as well as a different sequence of secretagogues). In spite of the well-accepted fact that both methods give valid results, as summarized elegantly by JP Clancy in his editorial [3], Sousa and colleagues felt it necessary to discredit the work of their competitors with the following phrase in the introductory section: “Assessment of CFTR (dys)function in native colonic epithelia ex vivo, as we previously reported, constitutes a good approach to this end [17,18]. However, since those data were reported, other groups have investigated the abnormalities in electrogenic Cl- secretion in the intestinal epithelium of CF patients using Ussing chamber measurements by different protocols [19–21]. Unfortunately, the final composite parameter used by some results from a combination of experimental readouts not all relying on direct measurement of CFTR-mediated Cl- secretion, thus leading to conflicting results and precluding good correlations with clinical symptoms [19–21].” This statement is not backed up by a systematic and scientific comparison of both protocols and certainly deserves a clarification for the readership of The Journal who may not be familiar with the methodical details of the two different ICM protocols. Hence, I would like to clarify with this correspondence:

1. Sousa and colleagues imply that the technique described by Mall and colleagues in 2004 [4,5], describing one of the two ICM protocols, precede the use of the open-circuit ICM protocol with the secretagogue sequence as described in 2006 and 2010 [2,6,7]. While this is technically correct, the data generated by van Barneveld et al [6,7] and Derichs et al [2] rely on the method developed in the early 1990s for ICM diagnosis in The Netherlands [8,9]. A consensus was attempted around the turn of the millennium, but as none could be reached, two protocols for ICM were published in the Journal of Cystic Fibrosis in 2004 ([4], quoted by Sousa and colleagues, and [10], not quoted by Sousa and colleagues).
In summary and clarification: the protocol used by van Barneveld et al [6,7] and by Derichs et al [2] was established well before 2004 [8,9] and has not been used SINCE, but well BEFORE Mall and colleagues [4,5] described their ICM protocol.

2. Sousa and colleagues [1] state that “the final composite parameter used by some results from a combination of experimental readouts not all relying on direct measurement of CFTR-mediated Cl- secretion, thus leading to conflicting results and precluding good correlations with clinical symptoms.” This grammatically arkwardly worded sentence likely intends to comment on the mode of data evaluation undertaken when interpreting the short-circuit ICM measurements [10]. Indeed, the short-circuit ICM protocol [10] relies on the interpretation of a biphasic response to carbachol in biopsies from cystic fibrosis individuals which is likely referred to in the phrasing by Sousa et al [1] of being “a combination of experimental readouts not all relying on direct measurement of CFTR-mediated Cl- secretion”. However, this is not a combination of “two experimental readouts”: Cholinergic stimulation by carbachol increases the intracellular Ca2+ concentration, causes a basolateral Ca2+-dependent K+ efflux, which in turn drives the apical Cl- secretion (please see [11] for a more detailed description of the principle of the assay). In the CF condition which is characterized by impaired or absent CFTR, the basolateral K+ efflux is visualized under short-circuit conditions [consensus protocol in 10], but not under open-circuit conditions [consensus protocol in 4]. In addition to this indirect pathway to a chloride secretory response, carbachol directly activates CFTR in the apical membrane by increasing the formation of diacylglycerol, thus stimulating the protein kinase C–dependent signaling pathway.
In summary and clarification: any ICM protocol that uses carbachol for stimulation of a chloride secretory response detects CFTR function mostly through an indirect pathway by increasing intracellular Ca2+ concentration. In other words, the protocol used by van Barneveld et al [6,7] and by Derichs et al [2] do NOT rely on “a combination of experimental readouts not all relying on direct measurement of CFTR-mediated Cl- secretion”, but they DO make use of the underlying biological principle by visualizing the basolateral K+ efflux in addition to an apical chloride secretory signal. The presence of a basolateral K+ secretory response has always been the hallmark of CF diagnosis by ICM by means of the short-circuit ICM protocol in diagnostic cases with severe pancreatic insufficient CF [2,8,9,10,11], mild pancreatic sufficient CF [2,8,9,10], patients homozygous for rare CFTR mutation genotypes [12] and cases with unclear diagnosis [2]. Among all cases tackled within these references [2,8,9,10,11,12] NO evidence of “conflicting results” or “ preclusion of good correlations with clinical symptoms” has been noticed. Quite the opposite is true: a correlation of ICM phenotype and CF disease severity has been noted a decade ago [13] and this has been successfully used to characterize inherited modifiers of CF disease [14].

3. We have yet to see a systematic approach in consistency with good scientific practice to answer the question which of the two protocols serves best as a measure of CFTR function – open-circuit or short circuit, and which secretagogue sequence will be optimal to quantify CFTR function. Until there is a systematic comparison of both assays, it may be best to judge both methods as outlined by JP Clancy in 2010 [3]: “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).”

PD Dr. rer. nat. Frauke Stanke
Department of Pediatric Pneumology and Neonatology
Hannover Medical School
Hannover, Germany
mekus.frauke@mh-hannover.de

[1] Sousa et al PLOSone 2012
[2] Derichs N, Sanz J, Von Kanel T, Stolpe C, Zapf A, et al. (2010) Intestinal current measurement for diagnostic classification of patients with questionable cystic fibrosis: validation and reference data. Thorax 65: 594–599.
[3] Diagnosing cystic fibrosis in patients with non-diagnostic results: the case for intestinal current measurements. Clancy JP. Thorax. 2010 Jul;65(7):575-6.
[4] Mall M, Hirtz S, Gonska T, Kunzelmann K (2004) Assessment of CFTR function in rectal biopsies for the diagnosis of cystic fibrosis. J Cyst Fibros 3 (S2): 165–169.
[5] Hirtz S, Gonska T, Seydewitz HH, Thomas J, Greiner P, et al. (2004) CFTR Clchannel function in native human colon correlates with the genotype and phenotype in cystic fibrosis. Gastroenterology 127: 1085–1095.
[6] van Barneveld A, Stanke F, Ballmann M, Naim HY, Tümmler B (2006) Ex vivo biochemical analysis of CFTR in human rectal biopsies. Biochim Biophys Acta 1762: 393–397.
[7] van Barneveld A, Stanke F, Tamm S, Siebert B, Brandes G, et al. (2010) Functional analysis of F508del CFTR in native human colon. Biochim Biophys Acta 1802: 1062–1069.
[8] 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.
[9] 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.
[10] De Jonge HR, Ballmann M, Veeze H, Bronsveld I, Stanke F, Tümmler B, Sinaasappel M. Ex vivo CF diagnosis by intestinal current measurements (ICM) in small aperture, circulating Ussing chambers. J Cyst Fibros. 2004 Aug;3 Suppl 2:159-63
[11] Bronsveld I, Mekus F, Bijman J, Ballmann M, Greipel J, Hundrieser J, Halley DJ, Laabs U, Busche R, De Jonge HR, Tümmler B, Veeze HJ. Residual chloride secretion in intestinal tissue of deltaF508 homozygous twins and siblings with cystic fibrosis. The European CF Twin and Sibling Study Consortium. Gastroenterology. 2000 Jul;119(1):32-40.
[12] Stanke F, Ballmann M, Bronsveld I, Dörk T, Gallati S, Laabs U, Derichs N, Ritzka M, Posselt HG, Harms HK, Griese M, Blau H, Mastella G, Bijman J, Veeze H, Tümmler B. Diversity of the basic defect of homozygous CFTR mutation genotypes in humans. J Med Genet. 2008 Jan;45(1):47-54.
[13] Bronsveld I, Mekus F, Bijman J, Ballmann M, de Jonge HR, Laabs U, Halley DJ, Ellemunter H, Mastella G, Thomas S, Veeze HJ, Tümmler B. Chloride conductance and genetic background modulate the cystic fibrosis phenotype of Delta F508 homozygous twins and siblings. J Clin Invest. 2001 Dec;108(11):1705-15.
[14] Stanke F, Becker T, Kumar V, Hedtfeld S, Becker C, Cuppens H, Tamm S, Yarden J, Laabs U, Siebert B, Fernandez L, Macek M Jr, Radojkovic D, Ballmann M, Greipel J, Cassiman JJ, Wienker TF, Tümmler B. Genes that determine immunology and inflammation modify the basic defect of impaired ion conductance in cystic fibrosis epithelia. J Med Genet. 2011 Jan;48(1):24-31.