Australasian guideline for the performance of sweat chloride testing 3rd edition: to support cystic fibrosis screening, diagnosis and monitoring

Guideline development group

The AACB-RCPAQAP Inborn Errors Advisory Committee (IEAC) (previously known as the Sweat Test Working Party) developed guidelines for sweat testing; originally in 2006 and most recently in 2017 [6], 8]. In 2024 this Committee consisted of all authors listed in this publication, with representation of paediatric clinical scientists (LG, RG, AM), pathologists (NH, EL, CS), RCPAQAP scientists (NR, PG) and a specialist in respiratory medicine (JM).

The AACB-RCPAQAP IEAC (via JM) and the Cystic Fibrosis Special Interest group (Dr. Simone Visser and Dr. Geshani Jayasuriya) of the Thoracic Society of Australia and New Zealand (TSANZ) have worked closely together to reconsider the clinical decision limits for sweat chloride.

Process for guideline update

In 2024 the IEAC decided there were sufficient changes to practice warranting the introduction of a revision of the guideline that was fit for purpose to support Australasian (and other regions) laboratories and clinicians.

The process for review is provided in Figure 1 and consisted of:

1. Meeting of CF special interest group of TSANZ to discuss the evidence and clinical practice for sweat chloride clinical decision limits (March 2024). This included both adult and paediatric CF physicians. There was consensus of this group that the USCFF and ECFS recommendations were routinely followed by CF clinicians and should be adopted in Australia.

2. Tabulation of other recent English language guidelines (Supplementary Table 1) [3], [4], [5], [6], [[8], [9], [10], [11], [12], [13].

3. Reviewing the 2nd edition guideline [6], performing a gap analysis, mapping proposed changes and using a traffic light system for the IEAC members to highlight if full agreement was reached. This process continued until all recommendations were fully endorsed.

4. Following an initial presentation of the draft recommendations at the AACB-RCPA Harmonisation Workshop (25 July 2024, Sydney Australia), an open online invitation to laboratory professionals was provided via the AACB website, and direct announcement to the RCPAQAP IEM program participants and the IFCC Committee-Emerging Technologies in Paediatric Laboratory Medicine (C-ETPLM) requesting feedback of the guideline table (Table 2) in the fourth quarter of 2024.

5. A post feedback review was conducted in February 2025 by IEAC.

Rationale for change of clinical decision limits

The change to a single intermediate clinical decision limit of 30–59 mmol/L was discussed by the IEAC, with the following points raised:

1. CF is not defined by a laboratory testing definition, but by a clinical definition that incorporates the sweat chloride test as one critical component in the decisional calculus;

2. For sweat chloride interpretation, we have moved away from laboratory-based reference intervals and re-cast them as clinical decision limits that have been adopted by clinical professional societies such as ECFS and US CFF [4], 5];

3. The change to a single intermediate decision limit for all patients simplifies the interpretation and decision pathway for clinical management;

4. All laboratories adopting the same set of decision limits provides harmonisation and is best practice for patient care; and

5. CF clinicians under the auspices of the CF Special Interest Group of the TSANZ support the 30–59 mmol/L intermediate decision limit.

Figure 1:

Overview of sweat test guideline review process. The formal consultation period opened on 7th October 2024 for feedback and closed on Friday 20th December 2024. AACB, Australasian Association for Clinical Biochemistry and Laboratory Medicine; RCPAQAP, Royal College of Pathologists of Australasia Quality Assurance Programs; NATA, National Association of Testing Authorities; IEAC, Inborn Errors Advisory Committee; IFCC C-ETPLM, International Federation of Clinical Chemistry and Laboratory Medicine Committee-Emerging Technologies in Pediatric Laboratory Medicine.

Pre analytical

The pre-analytical component incorporates seven key recommendations focused on patients and professionals.

Commonly a sweat chloride test will be requested for symptomatic patients or where there is a family history. In addition, in regions where newborn bloodspot screening (NBS) for CF occurs (including all of Australia and New Zealand), a sweat chloride test will routinely be ordered following a positive screening result as part of the diagnostic workup. Within the recommendations there are links to an example patient information sheet and a patient orientated video of the sweat testing process [14]. These documents help provide assurance for parents with a child about to undergo the sweat test.

Ongoing competency of sweat test collection team members is a key consideration in these recommendations. Sweat collection requires a standardised series of steps, starting with cleaning of the skin on the flexor surface of either forearm, placing a pilocarpine gel (or soaked pad) attached to electrodes on the arm, stimulation at 1.5–4 mA for 5 min, removal of the device and placement of the sweat collector on the arm for up to 30 min [9], 10]. At the end of this time the sweat sample is removed, and the minimum volume (or weight) checked. The minimum sweat volume for analysis is based on the rate of sweat secretion greater than 1 g/m²/min which equates to 18 μL of sweat collected in 30 min (or 15 μL in 25 min) [9], 10]. This process is well documented, and whilst many laboratories have moved to the coil tubing method, the principle is consistent with that developed by Gibson and Cooke in 1959 [26] and a multimedia training resource is freely available for health professionals [15].

Analytical

The analytical component incorporates six key recommendations focused on the laboratory processes.

The timeliness and sample handling post sweat collection are considered under analytical, because this is an area that the sweat testing laboratory has direct responsibility for validating the processes. From a patient and clinical perspective, the timeliness of analysis and reporting is important to reduce the anxiety associated with an undetermined diagnosis of CF. Ideally, the sweat test analysis should be undertaken on the same day of analysis, however, in some centres, there are logistic issues that will mean that this is not always feasible. Considering a patient-focused approach, the total testing process should minimise turnaround time as much as practicable.

Sweat collection usually occurs outside of the analytic laboratory site. The distance between collection and analysis can range from being in the same building as the laboratory to remote collection at sites hundreds of kilometres away. In considering transport, and storage, an important consideration is maintaining the integrity of the sweat sample immediately following collection to the start of analysis. There is some evidence that outlines appropriate transport and storage for sweat test samples collected remotely [16]. However, these processes should be evaluated by each laboratory to account for local circumstances.

There continues to be an evolving appreciation of the role of the sweat chloride test in the diagnosis and management of CF. This evolution is seen in successive guideline revisions, such as the removal of sodium from recommendations and the inclusion of considerations for remote sample collection [6]. Most recently, the RCPAQAP for sweat testing has removed conductivity analysis from its program because of a decline (to zero) in participating Australasian laboratories offering this test [27]. This reflects a change in practice, and a likely improvement in workflow with re-direction directly to sweat chloride testing.

Inductively coupled plasma mass spectrometry (ICP-MS) is the reference measurement procedure (RMP) for sweat chloride testing listed in the Joint Committee for Traceability in Laboratory Medicine (JCTLM) database [18], 19]. However, ICP-MS is not recognised in some other guidelines. For reference, in 2025 the RCPAQAP participation demonstrates that four out of the 26 participating laboratories use ICP-MS technology as their routine methodology, while the remaining laboratories employ other techniques such as Coulometry, IC-HPLC (ion chromatography-high-performance liquid chromatography) and colourimetry.

Irrespective of the method of chloride measurement, individual sweat chloride samples must NOT be pooled for analysis and samples <15 μL SHOULD NOT be analysed and MUST NOT be reported. It is the opinion of the IEAC that reporting the collection volume on the final report is useful as it provides a level of certainty that the laboratory has not deviated from this criterial sweat collection rate volume requirement.

Post-analytical

The post-analytical component of sweat testing incorporates three key recommendations focused on the reporting of the sweat chloride results and overall auditing of the total testing process.

Since the publication of the last guideline, it has become apparent that there is an increasing number of people with CF or CFTR related disease who have sweat chloride values between 30 and 39 mmol/L (as distinct from the previous lower limit of ≤40 mmol/L for the intermediate lower level); Figure 3. This is because of greater recognition by clinicians of the wide phenotype of CF and information generated by increasingly available CFTR gene analysis by next-generation sequencing, Multiplex Ligation-dependent Probe Amplification (MLPA) for deletions, and expansion to test for deep intronic variants that may be associated with CF or CFTR related diseases. Furthermore, the availability of highly effective CFTR modulators, such as the combination elexacaftror-tezacaftor-ivacaftor (ETI, marketed as Trikafta^®, in Australia and New Zealand, Vertex Pharmaceuticals) has meant that there are therapeutic options for people with troublesome disease mediated by CFTR.

Figure 3:

Relationship between sweat chloride concentrations and CF. (A) Information from the 2023 Cystic Fibrosis Foundation annual report, report median sweat chloride values for patient homozygous F508del have remained consistent over time, whereas for patients heterozygous for F508del, the median sweat chloride has decreased. This is likely a reflection of more patients with residual chloride function mutations being diagnosed with CF [28]. Reproduced with permission from the Cystic Fibrosis Foundation patient registry, 2023 annual data report. Bethesda, Maryland. ©2024 Cystic Fibrosis Foundation [28]. Reproduced with permission from the Cystic Fibrosis Foundation patient registry, 2023 annual data report. Bethesda, Maryland. ©2024 Cystic Fibrosis Foundation. (B) Relationship of sweat chloride between healthy adults and those with CF and CFTR disease.

Since it is clear that CF is understood as a spectrum of conditions relating to CFTR dysfunction, a traditional reference interval approach to the diagnosis of CF no longer has legitimacy. As such, the IEAC now refers to clinical decision-limits rather than reference intervals [29]. The clinical decision-limit still does not resolve the problem of the considerable overlap between the concentrations of sweat chloride for healthy (non-carrier) people and those with CFTR mediated disease, but this is best dealt with by a recommendation that sweat testing laboratories report intermediate results with a clear recommendation to consult a CF physician; Figure 4 [1]. In the face of an intermediate result, the CF physician will consider all the information needed to make a diagnosis of CF, synthesising a detailed CF phenotype with CFTR genetic information and the sweat chloride concentration.

Figure 4:

Interpretation of sweat chloride test results with one set of decision limits applied across all ages.

Finally, clinicians should be aware of the variation of the sweat chloride measurement which is partly clinical and partly due to measurement. There is no way of assessing the clinical variation, except for ensuring patients are well hydrated before the test and the test is collected in a standard way according to guidelines. However, CF is a clinical diagnosis and therefore the laboratory’s responsibility is to provide an appropriate report that recommends follow-up by a physician experienced in the diagnosis of CF for clinical evaluation.