Sweat Testing

Specimen Collection

The Cystic Fibrosis Foundation (CFF) recommends that a trained technician perform a sweat test at a CFF-accredited care center. The test is typically conducted on the patient’s arm or leg and involves iontophoresis of pilocarpine to stimulate sweat production by the sweat glands. Sweat may be collected using gauze, filter paper, or a macroduct coil.

Since sweat chloride levels may be transiently elevated within the first 24 hours of life, the CFF clinical care guidelines recommend performing sweat testing 48 hours after birth or later. Two samples are collected to account for the test’s variability and the possibility of an insufficient sample. Pilocarpine iontophoresis is performed for 5 minutes, followed by sweat collection for no more than 30 minutes. The stimulated area must be 2 in x 2 in when using gauze or filter paper. For the gauze method, a minimum of 75 mg of sweat is required, while the macroduct method requires a minimum sample volume of 15 μL.[6]

Procedures

The clinician performs pilocarpine iontophoresis on the patient’s skin. In the original Gibson-Cooke method, 2 electrodes—one covered with gauze soaked in pilocarpine and the other with deionized water—are placed on the patient’s arm or leg. A small, painless electrical current is applied for 5 minutes to stimulate sweat production. Afterward, sweat is collected using gauze, filter paper, or a Macroduct coil. The sweat collection time should not exceed 30 minutes.

Using the Gibson-Cooke method for QPIT, a minimum of 75 mg of sweat is required for adequate testing. However, most centers in the U.S. now use macroduct coil collection, which requires only 15 μL of sweat for analysis. Sweat should not be collected from multiple sites during the same test, as the chloride content depends on the sweating rate. The collected sample is then sent to the laboratory for chloride quantification.[7]

The Centers for Disease Control and Prevention does not classify sweat as a potentially infectious material unless it is visibly contaminated with blood. However, laboratory personnel should follow standard precautions as they would with any other body fluid. All equipment used for iontophoresis must be disinfected between patients in accordance with the institution’s infection control policies. Disinfectants should not contain bleach, as it can contaminate the sweat sample.[8]

Indications

Indications for the sweat test include suspicion of cystic fibrosis, either based on a positive newborn screening test or the presence of clinical features suggestive of the condition. The sweat test is considered the gold standard for confirmation. Cystic fibrosis genotyping is recommended to guide mutation-specific therapy in cases where sweat testing yields borderline results or is not technically feasible (eg, due to severe eczema). The CFF recommends genotyping for all patients.

In the U.S., newborn screening involves testing for elevated levels of immunoreactive trypsinogen (IRT). If elevated, the screening may reflex to a DNA test for known mutations of the CFTR gene, depending on state-specific protocols. A child with a positive newborn screening result will require confirmatory sweat testing at a cystic fibrosis center.

A referral to a cystic fibrosis center for sweat testing is indicated in children clinically suspected of having cystic fibrosis. Patients with cystic fibrosis may present with a wide range of symptoms. Neonates may show signs of meconium ileus, while young children may experience pulmonary complications such as recurrent pneumonia, upper respiratory infections, wheezing, and coughing. Gastrointestinal symptoms include failure to thrive, malabsorptive stools, and recurrent abdominal pain. Sweat testing may also serve as a surrogate biomarker for evaluating CFTR function. The results help clinicians assess the effects of CFTR-modulating drugs like ivacaftor.[9]

Potential Diagnosis

An elevated sweat chloride level (≥ 60 mmol/L) is diagnostic of cystic fibrosis, while sweat chloride levels below 29 mmol/L are considered normal. Levels ranging from 30 to 59 mmol/L are classified as borderline, necessitating a repeat sweat test or further testing. This range may indicate that the patient is a heterozygous carrier, and sweat testing is unreliable for detecting the condition in this group. The result of a positive newborn screen should be interpreted in conjunction with the sweat test to diagnose cystic fibrosis reliably. Additionally, certain known mutations associated with cystic fibrosis do not cause elevated sweat chloride levels, so sweat testing may not provide clarity in these cases.

Patients with a positive newborn screen and a negative sweat test may receive diagnoses other than cystic fibrosis, such as cystic fibrosis-related metabolic syndrome (CRMS) or cystic fibrosis screen-positive, inconclusive diagnosis (CFSPID). A normal sweat chloride test result does not exclude the diagnosis. In these cases, the clinician should consider genotyping or other diagnostic studies, such as nasal membrane potential difference, intestinal current measurement, semen analysis, or assessment of pancreatic function, particularly when cystic fibrosis is highly suspected.

Normal and Critical Findings

The sweat test results should be interpreted according to the following guide:

• Less than 30 mmol/L: Cystic fibrosis is unlikely.
• 30 to 59 mmol/L: Cystic fibrosis is possible; further testing may be required.
• 60 or greater mmol/L: Diagnostic of cystic fibrosis.[10]

Sweat chloride levels in patients without cystic fibrosis should be less than 30 mmol/L. Values of 60 mmol/L or greater are diagnostic of cystic fibrosis. Intermediate values, ranging from 30 to 59 mmol/L, are elevated but not diagnostic. Cystic fibrosis is possible in these patients, but repeat or alternative testing is necessary.

For patients with intermediate sweat chloride levels (30-59 mmol/L), genetic testing can help confirm or exclude the diagnosis. If 2 cystic fibrosis-causing mutations are identified on separate chromosomes, no further diagnostic testing is necessary, and the condition is confirmed. In patients without cystic fibrosis-causing mutations, no further testing is needed, as cystic fibrosis is unlikely. For patients with undefined CFTR mutations or mutations of varying clinical significance, the clinician should pursue additional testing if suggestive clinical features are present. Assessments may include nasal potential difference (NPD), intestinal current measurement (ICM), semen analysis, and pancreatic function testing.

Interfering Factors

The most common cause of falsely elevated sweat chloride levels is evaporation. Testing procedures should be carefully followed to minimize the risk of evaporation and contamination. Sweat samples should ideally be analyzed on the same day they are collected.[11] However, the laboratory can store the samples if necessary. Sweat collected on gauze should be reweighed immediately and may be stored in a tightly closed container at 4°C for up to 72 hours.[12]

False positives are rare, but sweat chloride levels may be falsely elevated due to external factors and underlying pathologic conditions, including the following:

• Improper testing technique
• Atopic dermatitis
• Anorexia nervosa
• Untreated adrenal insufficiency
• Glycogen storage disease
• Panhypopituitarism
• Hereditary nephrogenic diabetes insipidus
• Untreated hypothyroidism
• Pancreatitis
• Malnutrition
• Mucopolysaccharidosis
• Ectodermal dysplasia
• Prostaglandin E1 infusion

Newborns may not produce enough sweat and may need to wait until later in infancy for an adequate quantity to be collected. Infants with a positive newborn screening result can undergo testing as early as 2 days of life. However, the CFF recommends waiting until the child is 10 days old. For premature infants, testing should be delayed until they weigh at least 2 kilograms and have a corrected gestational age of more than 36 weeks, if possible.[13]

To increase the chances of obtaining an adequate sample, sweat may be stimulated from 2 sites during a single encounter. If the laboratory collects sweat from 2 sites (bilateral testing), the collection is considered insufficient (quantity not sufficient or QNS) only if both sites are inadequate. The forearm is preferred over the inner thigh for sweat stimulation in patients of all ages, as it typically produces more sweat after iontophoresis.[14]

The quality of sweat collection can be maintained by ensuring a sufficient test volume to uphold competency and limiting testing personnel to a small group of well-trained individuals. All CFF-accredited cystic fibrosis centers in the U.S. must adhere to Clinical and Laboratory Standards Institute (CLSI)-C34-A2 guidelines for sweat collection and analysis.[15]

Laboratories should implement a comprehensive quality assurance program for sweat testing to ensure accurate and reliable results. Daily quality control should involve analyzing at least 2 control samples with different chloride concentrations alongside patient samples. These controls verify that the analytical methods are functioning correctly and producing consistent results, enabling the prompt identification and correction of any deviations.[16]

To further ensure accuracy, laboratories should participate in external proficiency testing programs, such as those offered by the College of American Pathologists (CAP). In these programs, laboratories analyze standardized samples and compare their results with those from other laboratories to ensure consistency and compliance with established standards.[17] By integrating both internal quality control and external proficiency testing into the quality assurance process, laboratories can continuously monitor and enhance all aspects of sweat testing, from sample collection to result interpretation.[18]

Patient Safety and Education

Sweat testing is one of the few clinical chemistry tests involving direct patient contact. The patient should be physiologically and nutritionally stable, well-hydrated, free of acute illness, and not receiving mineralocorticoids. Additionally, patients should avoid applying moisturizers to the skin before sweat collection.

Sweat testing with QPIT is a quick, painless, safe, and reliable method for diagnosing cystic fibrosis. The electrical stimulation is painless and does not cause discomfort. For safety, the iontophoretic current source must be battery-powered. Periodic inspections for current control and leakage should be performed by biomedical engineering, following the manufacturer’s recommendations. Possible adverse events during sweat collection include redness and itching at the stimulation site. Pilocarpine-induced urticaria is rare but has been reported.[19] Iontophoresis can burn the patient’s skin, but this complication is extremely rare.[20]

Testing is conducted only at CFF-accredited cystic fibrosis centers. If a positive result is obtained, the diagnosis may be confirmed by either collecting a second sample or performing genetic testing. Other methods of measuring CFTR function, such as assessing total osmolality, conductivity, or sodium and potassium levels in sweat, are not recommended for diagnosis.

Clinical Significance

If clinical suspicion arises after newborn genetic screening or assessment by a healthcare provider, a sweat test should be performed to confirm the diagnosis of cystic fibrosis. The sweat test is considered the gold standard for diagnosing cystic fibrosis and is used in conjunction with clinical findings and family history. Cystic fibrosis is a lifelong condition with significant healthcare implications. Given the advancing therapies for this genetic disorder, early diagnosis is essential to ensure timely therapeutic intervention and clinical monitoring. Patients with cystic fibrosis are living longer than ever, with the median age of survival exceeding 40 years in developed countries. Children diagnosed later in life are likely to have better outcomes due to residual CFTR function and a less severe phenotype.[21]