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Clin Biochem Rev. 2007 May; 28(2): 60–76.
PMCID: PMC1904422

The Limitations of Sweat Electrolyte Reference Intervals for the Diagnosis of Cystic Fibrosis: A Systematic Review


The sweat test has been used for more than 50 years for the diagnosis of cystic fibrosis (CF) and remains an important diagnostic test in the genomic era. The currently used reference intervals for sweat electrolytes are applied to all patients regardless of age or sex. We performed a systematic review to summarise the studies with published reference values of sweat electrolyte concentrations for the diagnosis of CF. The MEDLINE (from 1950), EMBASE (from 1980) and PubMed (from 1950) databases were searched for English language studies. An abstract was also found by hand-searching. The search generated 1136 articles that matched the search key terms. Of these, 17 studies that contained data on sweat electrolyte concentrations were included in the analysis. Among these, seven studies did not perform the sweat test in accordance with current international and Australian guidelines. Of the ten remaining studies, four reported both the sweat sodium and chloride concentrations and six reported sweat chloride concentration only. A major limitation of these studies was the subject selection. Most recruited patients with various medical conditions including respiratory diseases or undefined recruitment criteria, whilst some did not report the subjects’ age and some had small subject numbers. Only one study performed mutation analysis to determine carrier status. No study used appropriate statistical analysis to develop a sweat chloride reference interval. The literature review yielded no studies that reliably developed reference intervals for sweat electrolyte concentrations. The limitations of the studies highlight the need for reliable age-related reference intervals for sweat electrolyte concentrations in healthy subjects.


CF is the most common autosomal recessive disorder among Caucasians in Australia with an incidence of 1 in 2500.1 The characteristic clinical features of CF are suppurative lung disease, pancreatic insufficiency, neonatal bowel obstruction (meconium ileus), multifocal biliary cirrhosis, absent vas deferens and high sweat electrolytes.2 Prior to the discovery of the CF transmembrane conductance regulator (CFTR) gene and the mutations responsible for CF, the diagnosis was based on clinical features and the measurement of elevated sweat electrolyte concentrations.2 The CFTR gene encodes a cAMP-regulated chloride channel which regulates chloride transport at the apical membrane of epithelial surfaces such as the airways, pancreatic ducts, biliary tree and sweat duct.2 In the sweat ducts, CFTR regulates chloride reabsorption and this forms the basis of the sweat test. Since the discovery of the CFTR gene in 1989, it has been possible to use gene mutation analysis as an alternative to sweat testing for the diagnosis of CF.35 However, more than 1200 mutations and polymorphisms have been identified6 and it is currently not feasible to routinely test for more than 20–30 of them. Some of the mutations are associated with a mild phenotype and this has increased the complexity of the diagnosis of CF. The sweat test is a measure of CFTR function and for this reason remains a valuable test for the diagnosis of CF, even in the genomic era.7

Analysis of sweat electrolytes has been used for the diagnosis of CF for more than 50 years.8,9 The first standardised methodology for sweat collection was introduced by Gibson and Cooke in 1959.9 Their method described the stimulation of muscarinic receptors by the application of pilocarpine to a local area of skin by iontophoresis and the subsequent collection of the sweat onto filter paper. A later advance on this technique was the development of the Wescor Macroduct collection system (www.wescor.com, Helena Laboratories, Mt Waverley, Victoria) in 1983.1012 The collection and analysis of the sweat sample is recognised as being technically demanding and subject to a number of pre-analytical and analytical limitations.1316 Recently international and Australian guidelines for the performance of the sweat test have been published with the common aim of further standardising both the collection and analytical components of the sweat test to improve accuracy.1315 However, these guidelines do not discuss the limitations of the individual studies upon which the recommended reference intervals are based.

Currently the universally accepted reference intervals for sweat chloride concentrations are: >60 mmol/L is considered diagnostic of CF; 40–60 mmol/L borderline; and <40 mmol/L normal.1315 Sweat sodium may be measured in addition to chloride for quality control purposes, since there is usually little difference between sweat sodium and chloride concentrations.1315 These reference intervals are applied to all patients regardless of age or sex. The aim of this review is to critically evaluate the published studies that include reference intervals for sweat electrolyte concentrations.


We performed a systematic review of all English language studies that included published reference values for sweat chloride and/or sodium concentrations in subjects with and without CF. The following databases were searched, OLD MEDLINE (1950 to 1965) and MEDLINE (1966 to 8 November 2006), EMBASE (1980 to 8 November 2006) and PubMed (1950 to 8 November 2006). The key words for the database search were sweat, electrolyte, pancreas, chloride, sodium, cystic fibrosis and sweat test (Figure). The titles and abstracts of the articles were screened (AM). We retrieved the full paper of studies that contained sweat electrolyte concentrations in non-CF and CF subjects. Reference lists of these articles were also searched for missing papers. The Australian and UK guidelines for the performance of the sweat test were also reviewed for additional data.15,17 In addition one abstract was found by hand searching and the author of this abstract was contacted to provide missing information. The relevant papers were reviewed by all three authors and information was collected on criteria which included subject selection (age, medical history, sample size and use of CFTR gene mutation analysis); sweat test methodology (sweat stimulation, collection and analysis), and the reporting of the sweat chloride and sodium concentrations. For this review we used the criteria described in 1994 by Solberg who defines an adequate sample size as one “consisting of at least 40 subjects.”18

Diagrammatic representation of the search methodology and inclusion criteria for the systematic review.


A total of 1135 articles matched the search terms for the respective databases: 60 articles in OLD MEDLINE, 345 in MEDLINE, 151 in EMBASE and 579 in the PubMed database (Figure). From these 1135 articles, 16 articles were found to be relevant.8,9,1932 An additional three studies which only presented data for the CF population were identified but did not fit the inclusion criteria of electrolyte concentrations on subjects with and without CF and thus were excluded.3335 One abstract, which was presented at the International Congress of Clinical Chemistry in 1993, was also included.36 In total there were 17 studies included in this review.

The sweat test methodology used in seven of the 17 identified studies8,1923,25 did not meet the accepted criteria for the performance of the sweat test as determined by the recently published national and international guidelines.1315 These seven studies include four that did not use pilocarpine iontophoresis to stimulate the sweat glands8, 1921 and three that did not report the mean sweat chloride concentration.22,23,25 The details of these studies are presented in Table 1.

Table 1
Review of the studies that did not comply with the guidelines for the performance of sweat testing.

Of the remaining 10 studies that used standard pilocarpine iontophoresis in the performance of the sweat test, four studies included results of both sweat chloride and sodium concentrations24,26,27,36 while six studies only reported sweat chloride concentrations.9,2832 Of the 10 studies, six reported mean and standard deviation sweat electrolyte concentrations.26,2932,36 The other four reported mean concentrations without other statistical parameters.9,24,27,28 The details of these studies are summarised in Table 2.

Table 2
Review of the studies that comply with international and Australian guidelines for the performance of sweat testing.

Of the 10 studies that used approved sweat testing methodology, four did not report the age of the subjects,9,26,29, 30 three studies were on subjects ≤15 years of age,28,31,36 one included adults 18 years of age and over27 and one included a wide age range of subjects.24 The age of the non-CF subjects in one of the papers was not clear.32 Of the 10 studies, three did not mention the recruitment criteria of the non-CF subjects.24, 29, 30 There were small numbers of non-CF subjects recruited in three studies,27,28,36 while five recruited only a small number of CF subjects.9, 2729, 36 Only one study performed CFTR gene mutation analysis to determine the carrier status of the non-CF group.31


The interpretation of sweat electrolyte concentrations has been used for the diagnosis of CF for over 50 years.8,9 As can happen in science, the details of the original experiments used to generate the data can be forgotten. The aim of this review was to understand the basis of the commonly accepted reference intervals by examining all the published studies on sweat electrolyte concentrations and to critically examine their validity in relation to the diagnosis of CF now that accepted methodology has been agreed on. To this effect we found 17 English language published studies generating reference intervals between 1950 and 1996.

From the 17 published studies, seven were deemed unacceptable, as they either did not use the currently accepted pilocarpine iontophoresis sweat test method or only reported sodium concentration.8,1923,25 Four of these studies were conducted before the development of the standardised pilocarpine iontophoresis sweat stimulation method1315 using inappropriate stimulation by heat,8 placing the patient in a plastic bag,21 or intradermal injection of the mecholyl hydrochloride.19,20 While the former two methods were simple,8, 21 they had major limitations such as the variability in the collection time (1–2 hours) required to obtain a sufficient sweat sample which raises the concern about evaporation and contamination while collecting the sweat. Other issues included infants becoming hyperpyrexic while placed in a plastic bag and there was a report of fatal heat stroke following this technique.9 In two studies sweat was stimulated by injection of mecholyl hydrochloride and the sweat was collected over the point of injection. This method did not become accepted as it was relatively painful for the subjects.19,20

It has been well documented that sweat chloride is most directly related to the abnormal function of CFTR and shows greater discrimination for the diagnosis of CF than sweat sodium.1315 For this reason, measuring sweat sodium alone is not recommended for the diagnosis of CF. Of the seven unacceptable studies, two analysed sweat sodium concentrations alone.22,25 In two other studies, sweat electrolyte reference intervals could not be identified as the mean sweat electrolyte concentrations were not stated.19,23 In one of these two studies, the results were tabulated with the number of subjects showing various sweat sodium levels19 and in another study the sweat sodium concentrations were graphically presented.23

There were 10 studies that used the standardised pilocarpine iontophoresis method for sweat collection.9,24,2632,36 However each of these studies suffered from significant limitations, including subject selection, non-specification of the age of the sample population, lack of CFTR gene mutation analysis, small sample size, and incorrect statistical methods employed to develop reference intervals. In some cases the reasons for these methodological flaws were historical, such as limited knowledge about the broader CF phenotype and inability to perform CFTR gene mutation analysis (pre 1989). In other examples, however, the investigators used samples of convenience that included patients referred for sweat tests or parents and siblings of CF patients, most of whom were likely to be carriers. Many of the studies employed inappropriate statistics, the commonest error being the use of mean values from non-parametric data. Due to the variations between the 10 studies, the sweat electrolyte concentrations generated from each of the studies were not comparable to each other and so we could not perform meta-analysis.

A principal limitation of the ten studies that did use the standardised methodology was the lack of a ‘healthy’ control group.9,24,2629,32 Subjects were recruited from hospital wards and clinics and also included subjects with a variety of medical conditions including respiratory illnesses that could have been CF.9,2729,32,36 Alternatively subjects were generated by referrals for a sweat test for clinical reasons9,24,26,32 or from infants that had a positive newborn screening test result.31 In three of the 10 studies the recruitment of non-CF control groups was not defined24,29,30,36 and in another five studies the non-CF subjects’ age was not mentioned.9,26,29,30,32 As our understanding of CF has developed, it is possible that phenotypes that were once not thought to be consistent with CF have now been included in the expanded clinical spectrum of CF. It is also possible that some of these non-CF subjects with diseases other than CF could have conditions that cause an elevation of sweat electrolyte concentrations.

Among these 10 studies, six were undertaken before the discovery of the CFTR gene in 1989.9,26,2830,32 However, even at that time the investigators would have been aware of the inheritance of CF. From the four studies that were undertaken after the availability of CFTR gene mutation analysis, only one study performed the CFTR gene mutation analysis (for the most common mutation ΔF508) to differentiate carriers from the non-CF control groups.31 No studies of subjects that included adolescents or adults performed CFTR gene mutation analysis. Therefore, some control individuals, many of whom were siblings or parents of CF patients, could have been carriers and may have skewed the results. This assertion is supported by the study in infants demonstrating that CF carriers had a mean sweat chloride concentration of one standard deviation above control subjects.31

The development of a reliable reference interval requires an adequate sample size. In 1994 Solberg recommended that the sample population should consist of at least 40 participants.18 Three of the 10 studies, had a small number of non-CF subjects to allow definite reference intervals to be stated.27,28,36 The remaining seven studies reported the sweat chloride or sodium concentrations as mean (± standard deviation) without mentioning the distribution of the data. We are now aware of the importance of the distribution, which determines the appropriate statistical analysis to be used. A general rule is that parametric analysis is used for normally distributed data and non-parametric analysis for data that are skewed,37 which is then followed by a series of step-by-step statistical calculations to develop 95% or 99% reference intervals.

Of the ten studies using acceptable methodology, the study of infants by Farrell and Koscik in 1996 is probably the most reliable study as it recruited a large number of subjects (n= 707), performed CFTR gene mutation analysis to determine the carrier status of non-CF subjects, and referred to the distribution of the sweat chloride concentrations.31 The authors of this study recommended that a sweat chloride concentration ≥40 mmol/L be used to distinguish infants with CF, which represented the mean plus three standard deviation values for the group of 128 CF heterozygote carriers identified by gene mutation analysis. Despite acknowledging that the sweat chloride concentration data were skewed in the heterozygote carrier group and in the healthy infants group, they still reported the results as mean (± standard deviation) concentration rather than median.

The question of reference intervals in relation to age was raised in seven of the 17 studies. These studies infer that sweat chloride and sodium concentrations increase with age.15,1720,22,31 The study by Doery and Huang in 1993 reported increasing sweat chloride and sodium concentrations with age in non-CF subjects, however they had only a small number of subjects in the 10+ year age group.36 This was the only study to take a systematic approach to sweat electrolytes with increasing age, but provides little information to guide clinicians requesting sweat tests in adolescents and adults. This is an increasing problem as the CF phenotype can include patients presenting beyond infancy and childhood. At The Royal Children’s Hospital (Melbourne, Victoria, Australia) 9% of the patients referred for a sweat test were older than 15 years in 2004 and 13% in 2005 (unpublished data). Accurate interpretation of sweat chloride and sodium concentrations in these patients requires knowledge of the age-related changes in sweat chloride and sodium concentrations.

In conclusion, we identified 17 studies over a 50 year period that reported sweat chloride with or without sodium concentrations for the diagnosis of CF. All 17 studies had limitations according to the current guidelines. Most studies did not include a ‘healthy’ control group, were performed prior to the availability of the CFTR gene mutation analysis and did not comply with the currently accepted sweat test method. This systematic review has revealed that there is no definitive study that quantitates the sweat chloride and sodium concentration in a truly normal population. Further work is required to re-establish sweat electrolyte reference intervals, using the current standardised sweat test, CFTR gene mutation analysis to exclude carriers, and correct statistical analyses in healthy participants with ages spanning from infancy to adulthood.


We gratefully acknowledge Angela Chiriano for critical reading of the manuscript.


Competing Interests: None declared.


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