Pediatric Pulmonology 49:106–117 (2014)
State of the Art Sixty-Five Years Since the New York Heat Wave: Advances in Sweat Testing for Cystic Fibrosis Jake T.B. Collie, BBioMedSc,1,2,3 R. John Massie, FRACP, PhD,3,4,5,6† Oliver A.H. Jones, PhD, MRACI CChem,1 Vicky A. LeGrys, Dr A, MT (ASCP),7 and Ronda F. Greaves, PhD, FFSc (RCPA)3,6,8*† Summary. The sweat test remains important as a diagnostic test for cystic fibrosis (CF) and has contributed greatly to our understanding of CF as a disease of epithelial electrolyte transport. The standardization of the sweat test, by Gibson and Cooke [Gibson and Cooke (1959) Pediatrics 1959;23:5], followed observations of excessive dehydration amongst patients with CF and confirmed the utility as a diagnostic test. Quantitative pilocarpine iontophoresis remains the gold standard for sweat induction, but there are a number of collection and analytical methods. The pathophysiology of electrolyte transport in sweat was described by Quinton [Quinton (1983) Nature 1983;301:421–422], and this complemented the developments in genetics that discovered the cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial-based electrolyte transport protein. Knowledge of CF has since increased rapidly and further developments in sweat testing include: new collection methods, further standardization of the technique with international recommendations and age related reference intervals. More recently, sweat chloride values have been used as proof of effect for the new drugs that activate CFTR. However, there remain issues with adherence to sweat test guidelines in many countries and there are gaps in our knowledge, including reference intervals for some age groups and stability of sweat samples in transport. Furthermore, modern methods of elemental quantification need to be explored as alternatives to the original analytical methods for sweat electrolyte measurement. The purpose of this review is therefore to describe the development of the sweat test and consider future directions. Pediatr Pulmonol. 2014; 49:106–117. ß 2013 Wiley Periodicals, Inc. 1 School of Applied Sciences, RMIT University, Melbourne, Victoria, Australia. 2
Dorevitch Pathology, Heidelberg, Victoria, Australia.
3
Murdoch Children’s Research Institute, Parkville, Victoria, Australia.
4
Department of Respiratory Medicine, Royal Children’s Hospital, Parkville, Victoria, Australia. 5 Department of Paediatrics, University of Melbourne, Melbourne, Victoria, Australia. 6 Australasian Association of Clinical Biochemists, Sweat Test Working Party, , Alexandria, New South Wales, Australia. 7 Department of Allied Health Sciences, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill North Carolina.
review article was conceived by Dr. Massie and Dr. Greaves. Dr. Massie aided in the literature search, reviewed and revised the manuscript, and approved the final manuscript as submitted. Ronda F. Greaves: The concept of this review article was conceived by Dr. Greaves and Dr. Massie. Dr. Greaves aided in the literature search, reviewed and revised the manuscript, and approved the final manuscript as submitted. Jake T.B. Collie: Mr Collie aided in the literature search, wrote the first and subsequent drafts of the review article, reviewed and revised the manuscript, and approved the final manuscript as submitted. Oliver A.H. Jones: Dr. Jones aided in the literature search, added his expertise in inductively coupled plasma mass spectrometry (ICP-MS) analysis of sweat, reviewed and revised the manuscript, and approved the final manuscript as submitted. Vicky A. LeGrys: Dr. LeGrys aided in the literature search and sourced the historical versions of the Clinical Laboratory Standards Institute Sweat Testing guidelines, critically reviewed the manuscript, and approved the final manuscript as submitted. †
http://www.aacb.asn.au/professionaldevelopment/sweat-testing.
School of Medical Sciences, RMIT University, Bundoora, Victoria, Australia.
Correspondence to: Ronda F. Greaves, PhD, FFSc (RCPA), School of Medical Sciences, RMIT University, PO Box 71, Bundoora, Vic, 3083, Australia. E-mail: [email protected]
Conflict of interest: None.
Received 11 April 2013; Accepted 2 September 2013.
Author contribution: This review consists of original material and has not been published elsewhere; except where duly referenced. All authors listed have made substantive intellectual contributions to this work, including the concept, design, and literature search. R. John Massie: the concept of this
DOI 10.1002/ppul.22945 Published online 19 November 2013 in Wiley Online Library (wileyonlinelibrary.com).
8
ß 2013 Wiley Periodicals, Inc.
Advances in Cystic Fibrosis Sweat Chloride Testing
107
Key words: cystic fibrosis; sweat test; sweat chloride methods; standardization; best practice guidelines; CFTR activation. Funding source: none reported
INTRODUCTION
Over the past 65 years there have been extraordinary advances in the care of patients with cystic fibrosis (CF). The key development that identified CF as a disease entity, separating it from other conditions causing malnutrition in children, was the sweat test. The availability of an accurate diagnostic test allowed the study of CF pathogenesis resulting in the identification of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The clinical manifestations of CF are now well understood and the first generation of therapies which aim to restore CFTR function have been developed. There have been significant advances in performance and interpretation of the sweat test, and it remains a key modality for the diagnosis of CF and has been a helpful proof of effect in the initial studies of therapies that restore CFTR function. It is therefore timely to examine the evolution of the sweat test and consider future developments. HOW THE SWEAT TEST BEGAN
In the summer of 1948 (10 years after Andersen’s landmark work identifying CF as a disease entity) several cases of dehydration, fever and shock were reported during the New York heat wave.1,2 Kessler and Andersen, and Di Sant’ Agnese, reported findings demonstrating that patients with fibrocystic disease of the pancreas (i.e., CF) were more susceptible to heat prostration.3,4 These observations led to the identification of abnormally high electrolyte concentrations in the sweat of these
ABBREVIATIONS: CF Cystic fibrosis CFTR Cystic fibrosis transmembrane conductance regulator QPIT Quantitative pilocarpine iontophoresis technique ISE Ion selective electrode CFF American Cystic Fibrosis Foundation ORCC Outwardly rectifying chloride channel CaCC Calcium activated chloride channel NCCLS National Committee for Clinical Laboratory Standards CLSI Clinical Laboratory Standards Institute CAP College of American Pathologists UK United Kingdom AUS Australia RCPA Royal College of Pathologists Australia QAP Quality Assurance Programs UK NEQAS United Kingdom National External Quality Assessment Scheme ICP-MS Inductively coupled plasma mass spectrometry
patients. Together, these were the first significant developments of what was to become the sweat test. Sweat Collection
The early techniques to induce sweat involved “thermal stress,” placing the patient in a heated room (908F/ 32.28C), for 1–2 hr.5 In 1955 a simpler method of thermal stress sweat collection was reported, with the patient wearing a plastic body bag tied loosely at the neck and covered in blankets for 60–90 min.6 This method was widely adopted although there were risks, including one reported fatality.6 Not surprisingly, this prompted a series of investigations into safer methods of sweat collection. Alternative strategies for detecting chloride in the skin through finger prints on plates impregnated with silver nitrate and potassium chromate was devised by Schwachman and Gahn.7 Similar methods were also reported by Webb, who placed filter paper soaked in silver nitrate between the fingers, and Gluck, who used a patch film test using the patient’s palm.8,9 Each of these tests relied on a color change when the silver nitrate reacted with the chloride in sweat. Although the silver nitrate tests were safer and simpler compared to the “thermal stress” tests, they were never fully validated and were rapidly replaced when the quantitative pilocarpine iontophoresis technique (QPIT) was developed. In 1959 Gibson and Cooke published the landmark method for sweat stimulation from eccrine sweat glands of the forearm, using topical application of pilocarpine through iontophoresis; the QPIT method.10 Pilocarpine is a parasympathomimetic alkaloid which acts on the cholinergic receptors by mimicking acetylcholine, a neurological transmitter. The charged pilocarpine is propelled transdermally by repulsive electromotive force using a small electrical current, that is, iontophoresis.11 This stimulates the muscarinic receptors on the eccrine sweat glands to induce sweat secretion.2,12 In the Gibson and Cooke QPIT method the sweat is collected onto gauze or filter paper. The collected sweat is then eluted from the collection material and analyzed using coulometry, colorimetry or ISE as a method for chloride and flame photometry for sodium. The Gibson and Cooke QPIT for sweat collection was not without its drawbacks. Electrode exposure, causing burns and blistering on the site of contact, were a known risk, although this was fundamentally due to technical errors from inexperienced sweat collectors exceeding the maximum electrical voltage or stimulation time.13,14 Over diagnosis of CF, due to high chloride results from poor sample collection as well as sample evaporation also Pediatric Pulmonology
108
Collie et al.
occurred.15,16 Despite these problems the Gibson and Cooke method remained the only recognized method of sweat collection for the next 25 years. In 1983 the Wescor (Logan, Utah) Macroduct system of sweat collection was introduced (See Fig. 1).17,18 This system restructured the conventional QPIT into a comparatively simplified collection method removing
the need for reagent preparation.19 The iontophoretic current passes through specially designed gel discs containing pilocarpine, which is delivered through the skin to the eccrine sweat glands by iontophoresis. The resulting sweat is collected into coiled capillary tubing. The sample is analyzed by a variety of techniques. The popularity of this system is largely attributed to its
Fig. 1. Gibson and Cooke and Wescor sweat collection consumables. A: Iontophroesis system with electrodes and straps (Wescor shown here). Wescor—B: Pilocarpine impregnated gel discs for sweat stimulation, C: Macroduct coil sweat collection devise, D: Macroduct collection tube. Gibson and Cooke—E: Gauze pad soaked in pilocarpine for sweat stimulation, F: Pre-weighed gauze pad for sweat collection, G: Pre-weighed storage pot containing collected sweat on gauze.
Pediatric Pulmonology
Advances in Cystic Fibrosis Sweat Chloride Testing
comparative automation of sweat stimulation and ease of use. The Wescor Macroduct was approved for sweat collection by the American Cystic Fibrosis Foundation (CFF) in 1990.18 Today, both the Gibson and Cooke QPIT and the Macroduct systems are recommended sweat collection methods for the investigation of CF and published comparison of these two systems report equivalent results.19–23 A significant difference between the Gibson and Cooke QPIT and Macroduct QPIT sweat collection systems relates to the sample matrix. The Gibson and Cooke QPIT sample is a diluted sample due to the need for elution from the collection padding. The Macroduct QPIT sample is collected directly into tubing and can be analyzed neat. The choice of collection technique therefore influences the method of analysis. Gibson and Cooke suggested reference intervals for sweat chloride (and sodium) which surprisingly, given the relative lack of rigour in collection of samples from healthy subjects, have stood the test of time. Further clinical contributions have been made through the development of reference intervals for most age groups.24,25 Through these developments, our understanding of CF has also changed with recognition that there is a spectrum of clinical manifestations relating to CFTR dysfunction and that the sweat test contributes to this understanding.26 THE SWEAT CHLORIDE TEST AS A MEASURE OF CFTR FUNCTION
The original observations of elevated sweat electrolytes in patients with CF had no recognized physiological basis
109
and seemed at odds with a disease that caused sticky mucous in the lungs, pancreatic ducts and gut. Observations from studies of the pancreatic ducts and nasal epithelium suggested the problem was one of electrolyte transport at epithelial surfaces. 27–29 However, it was analysis of the sweat ducts by Quinton30 that identified the basic defect to be specific to chloride ion transport. Knowledge of the electrolyte transport defect was fundamental in the efforts to discover the genetic basis of CF. In 1989, the CFTR protein and the common mutation associated with most cases of CF, was discovered on chromosome 7 (7q31.2).31–33 The structure of CFTR was confirmed as an epithelial electrolyte transport protein, predominantly for chloride but also regulating a separate sodium channel (ENaC). There are other chloride channels such as the outwardly rectifying chloride channel (ORCC) and the calcium activated chloride channel (CaCC) that are an alternative pathway for chloride transport. Normal CFTR function and the defect resulting from the common mutations are outlined in Figure 2. Following the original identification of the common CFTR mutation p.Phe508del (formerly DF508), other mutations were rapidly identified and there are now over 1,900 known mutations and polymorphisms.34 Nevertheless, most clinical laboratories test a limited number of the more common CFTR mutations. In addition CFTR sequencing is expensive and may yield sequence variations of unknown significance. For these reasons, genetic testing has not replaced the sweat chloride test as the major diagnostic tool for CF.35 This is reflected in the
Fig. 2. Diagram outlining the transportation of chloride and sodium through a normal, p. Phe508del and p.Gly551asp mutations of a sweat duct in the eccrine gland.
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Collie et al.
consensus statements for the diagnosis of CF which rely on a combination of recognized clinical features, CFTR gene mutations and the QPIT sweat chloride test. 36,37 Investigations of the first drug to successfully restore CFTR function of the sweat gland recognized the value of the sweat test to monitor this function.38,39 In patients with CF, with the CFTR mutation p.Gly551asp (formally G551D), the mean sweat chloride level reduced from a mean of 100.4–52.5 mmol/L confirming the biological effect of the drug ivacaftor (Vertex Pharmaceuticals, Cambridge, MA) activating sweat gland CFTR function.39,40 The precise mechanism of this action remains unknown and there is speculation regarding whether there was any organ specific restoration of CFTR function, other than the sweat gland, but the sweat test played an important role in determining that the efficacy of ivacaftor was due to improved CFTR function in patients with the Gly551Asp mutation.39,41 As such, the future role of the sweat test is likely to include the monitoring of treatment success and compliance, in addition to its traditional role in the investigation of CF. STANDARDIZATION OF THE SWEAT CHLORIDE TEST: WHERE ARE WE TODAY?
Through the development of guidelines, there is one agreed methodology for sweat collection, pilocarpine iontophoresis, and several methodologies for sweat analysis. Some of these variations include which analyte is to be measured, how it is analyzed and what reference intervals are applicable to the results. Recent reports indicate that there is still a distinct lack of laboratory standardization in most countries, which
appears to be due to poor adherence to published guidelines. 42–44 Guidelines
The need for standardization of the collection, analysis and reporting of the sweat test was addressed by the first National Committee for Clinical Laboratory Standards (NCCLS—now known as the Clinical Laboratory Standards Institute or CLSI) guideline in 1994.45 Since then there has been refinement of sweat test methodologies, the development of guidelines in other countries and updates of the CLSI guideline. The following discussion covers the English language guidelines but acknowledges that other non-English procedures, such as the French Guideline, have been published.46 (A definition of standardization is provided in Table 1.47) Following the first version of the CLSI guideline for the sweat chloride test in 1994, CLSI published updated guidelines in 2000 and again in 2009.23,45,48 The CFF produced a guideline of their own in 2007 which was based on the 2nd CLSI guideline and the College of American Pathologists (CAP) Laboratory Accreditation Program Inspection Checklist.49 The United Kingdom (UK) developed an evidence based guideline for sweat chloride testing in 2003 as did Australia (AUS) in 2006.20,21 A comparison of these guidelines, highlighting critical aspects for standardization, can be seen in Table 2. Today, apart from CFF and CAP accreditation, there is still limited demonstrated enforcement of compliance with the guidelines. Without compliance, inconsistency in sweat chloride testing between laboratories is evident. For example, an Italian survey uncovered inconsistencies, including sweat
TABLE 1— Definitions Related to Methods of Analysis for Sweat Chloride Testing
Terminology
Description
Addition dilution
‘In relation to the Sweat Test the term “Addition Dilution” refers to the addition of a known concentration (e.g., Standard of Control) to a sample in order to calculate a result which would otherwise be below the sensitivity of the method’ Determines the concentration by measuring the absorbance of a specific wavelength of light. The intensity of the colour is directly proportional to concentration Analytical chemistry technique that utilizes an electrolysis reaction to measure the changes in resistance to the current between electrodes. The concentration of the titrant is equivalent to the current generated Converts the activity of a specific ion dissolved in a solution into an electrical potential which is measured by a voltmeter Inductively Coupled Plasma Mass Spectrometry or ICP-MS is an analytical technique used for elemental determinations. It was commercially introduced in 1983 and combines a high-temperature ICP source with a mass spectrometer. The plasma source converts the atoms of the elements in the sample to ions that are then separated and detected by the mass spectrometer Standardization as a concept is the agreement of test results. In relation to the sweat test, the term “standardization” incorporates all aspects of the test from patient eligibility through to clinical interpretation. For the analysis of sweat chloride, standardization specifically refers to the establishment of traceability to higher order reference materials and measurement procedures with the outcome of agreement with established trueness of the result
Colorimetry Coulometry Ion selective electrode ICP-MS
Standardization
Pediatric Pulmonology
5 min Max 30 min No information available within guideline
Sweat conductivity
Sodium—Flame photometry
Stimulation time Collection time Storage
Screening method
Method of analysis
Current
Chloride—Titration with chloride meter
0.075 g—Gauze/Filter paper 15 ml—Microbore tubing 0.075 g—Gauze/Filter paper 15 ml—Microbore tubing 4 mA
Minimum weight/ volume
Minimum age/weight of patient
Gauze or filter paper low in sodium and chloride OR Microbore tubing >48 hr/No minimum weight
1994
Medium of collection
Year
CLSI 1st Edition
Chloride—Titration with chloride meter. Advice against ISE. Other methods acceptable if validated
Sodium—Flame photometry
Sweat conductivity, Sweat Sodium (flame photometry)
5 min Max 30 min No information available within guideline
4 mA
0.075 g—Gauze/Filter paper 15 ml—Microbore tubing
Gauze or filter paper low in sodium and chloride OR Microbore tubing >48 hr/No minimum weight
2000
CLSI 2nd Edition
Chloride—Colorimetry, Coulometry, and ISE
Sweat conductivity— Wescor equipment only and a CV of 48 hr/No minimum weight
2007
CFF
Washed filter paper or gauze OR Wescor Macroduct collectors >2 weeks of age/>3 kg
2006
Australia
(Continued)
Other methods acceptable if validated
5 min Max 30 min Stable for 72 hr. Gauze or filter paper in a vial. Microcentrifuge tubes Sample must not be stored in Microbore tubing Sweat Conductivity (outside of cystic fibrosis accredited centres only) Chloride—Titration with chloridemeter. Caution using ISE
2.5–4 mA
Gauze or filter paper low in sodium and chloride OR Microbore tubing 2 weeks of age/>2 kg OR 48 hr of age if clinically important and sufficient sweat collected 0.075 g - Gauze/Filter paper 15 mL—Microbore tubing
2009
CLSI 3rd Edition
TABLE 2— Comparison of Evidence Based English Language Guidelines for the Standardization of Sweat Testing for Use in the Investigation of Cystic Fibrosis by the Australian Sweat Test Working Party (AUS), UK Multi-Disciplinary Working Group (UK), Cystic Fibrosis Foundation (CFF), CLSI and Clinical Laboratory Standards Institute (CLSI - formally NCCLS)
Advances in Cystic Fibrosis Sweat Chloride Testing 111
Pediatric Pulmonology
30 mmol/L considered abnormal 60 mmol/L supports CF diagnosis >60 mmol/L supports CF diagnosis
40–60 mmol/L equivocal
60 mmol/L supports CF diagnosis >60 mmol/L supports CF diagnosis Sweat Chloride Reference Intervals
2007 2006 2003 2000 1994 Year
CFF Australia United Kingdom CLSI 2nd Edition CLSI 1st Edition
TABLE 2— (Continued)
2009
Collie et al. CLSI 3rd Edition
112
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collection times extending from
State of the Art Sixty-Five Years Since the New York Heat Wave: Advances in Sweat Testing for Cystic Fibrosis Jake T.B. Collie, BBioMedSc,1,2,3 R. John Massie, FRACP, PhD,3,4,5,6† Oliver A.H. Jones, PhD, MRACI CChem,1 Vicky A. LeGrys, Dr A, MT (ASCP),7 and Ronda F. Greaves, PhD, FFSc (RCPA)3,6,8*† Summary. The sweat test remains important as a diagnostic test for cystic fibrosis (CF) and has contributed greatly to our understanding of CF as a disease of epithelial electrolyte transport. The standardization of the sweat test, by Gibson and Cooke [Gibson and Cooke (1959) Pediatrics 1959;23:5], followed observations of excessive dehydration amongst patients with CF and confirmed the utility as a diagnostic test. Quantitative pilocarpine iontophoresis remains the gold standard for sweat induction, but there are a number of collection and analytical methods. The pathophysiology of electrolyte transport in sweat was described by Quinton [Quinton (1983) Nature 1983;301:421–422], and this complemented the developments in genetics that discovered the cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial-based electrolyte transport protein. Knowledge of CF has since increased rapidly and further developments in sweat testing include: new collection methods, further standardization of the technique with international recommendations and age related reference intervals. More recently, sweat chloride values have been used as proof of effect for the new drugs that activate CFTR. However, there remain issues with adherence to sweat test guidelines in many countries and there are gaps in our knowledge, including reference intervals for some age groups and stability of sweat samples in transport. Furthermore, modern methods of elemental quantification need to be explored as alternatives to the original analytical methods for sweat electrolyte measurement. The purpose of this review is therefore to describe the development of the sweat test and consider future directions. Pediatr Pulmonol. 2014; 49:106–117. ß 2013 Wiley Periodicals, Inc. 1 School of Applied Sciences, RMIT University, Melbourne, Victoria, Australia. 2
Dorevitch Pathology, Heidelberg, Victoria, Australia.
3
Murdoch Children’s Research Institute, Parkville, Victoria, Australia.
4
Department of Respiratory Medicine, Royal Children’s Hospital, Parkville, Victoria, Australia. 5 Department of Paediatrics, University of Melbourne, Melbourne, Victoria, Australia. 6 Australasian Association of Clinical Biochemists, Sweat Test Working Party, , Alexandria, New South Wales, Australia. 7 Department of Allied Health Sciences, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill North Carolina.
review article was conceived by Dr. Massie and Dr. Greaves. Dr. Massie aided in the literature search, reviewed and revised the manuscript, and approved the final manuscript as submitted. Ronda F. Greaves: The concept of this review article was conceived by Dr. Greaves and Dr. Massie. Dr. Greaves aided in the literature search, reviewed and revised the manuscript, and approved the final manuscript as submitted. Jake T.B. Collie: Mr Collie aided in the literature search, wrote the first and subsequent drafts of the review article, reviewed and revised the manuscript, and approved the final manuscript as submitted. Oliver A.H. Jones: Dr. Jones aided in the literature search, added his expertise in inductively coupled plasma mass spectrometry (ICP-MS) analysis of sweat, reviewed and revised the manuscript, and approved the final manuscript as submitted. Vicky A. LeGrys: Dr. LeGrys aided in the literature search and sourced the historical versions of the Clinical Laboratory Standards Institute Sweat Testing guidelines, critically reviewed the manuscript, and approved the final manuscript as submitted. †
http://www.aacb.asn.au/professionaldevelopment/sweat-testing.
School of Medical Sciences, RMIT University, Bundoora, Victoria, Australia.
Correspondence to: Ronda F. Greaves, PhD, FFSc (RCPA), School of Medical Sciences, RMIT University, PO Box 71, Bundoora, Vic, 3083, Australia. E-mail: [email protected]
Conflict of interest: None.
Received 11 April 2013; Accepted 2 September 2013.
Author contribution: This review consists of original material and has not been published elsewhere; except where duly referenced. All authors listed have made substantive intellectual contributions to this work, including the concept, design, and literature search. R. John Massie: the concept of this
DOI 10.1002/ppul.22945 Published online 19 November 2013 in Wiley Online Library (wileyonlinelibrary.com).
8
ß 2013 Wiley Periodicals, Inc.
Advances in Cystic Fibrosis Sweat Chloride Testing
107
Key words: cystic fibrosis; sweat test; sweat chloride methods; standardization; best practice guidelines; CFTR activation. Funding source: none reported
INTRODUCTION
Over the past 65 years there have been extraordinary advances in the care of patients with cystic fibrosis (CF). The key development that identified CF as a disease entity, separating it from other conditions causing malnutrition in children, was the sweat test. The availability of an accurate diagnostic test allowed the study of CF pathogenesis resulting in the identification of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The clinical manifestations of CF are now well understood and the first generation of therapies which aim to restore CFTR function have been developed. There have been significant advances in performance and interpretation of the sweat test, and it remains a key modality for the diagnosis of CF and has been a helpful proof of effect in the initial studies of therapies that restore CFTR function. It is therefore timely to examine the evolution of the sweat test and consider future developments. HOW THE SWEAT TEST BEGAN
In the summer of 1948 (10 years after Andersen’s landmark work identifying CF as a disease entity) several cases of dehydration, fever and shock were reported during the New York heat wave.1,2 Kessler and Andersen, and Di Sant’ Agnese, reported findings demonstrating that patients with fibrocystic disease of the pancreas (i.e., CF) were more susceptible to heat prostration.3,4 These observations led to the identification of abnormally high electrolyte concentrations in the sweat of these
ABBREVIATIONS: CF Cystic fibrosis CFTR Cystic fibrosis transmembrane conductance regulator QPIT Quantitative pilocarpine iontophoresis technique ISE Ion selective electrode CFF American Cystic Fibrosis Foundation ORCC Outwardly rectifying chloride channel CaCC Calcium activated chloride channel NCCLS National Committee for Clinical Laboratory Standards CLSI Clinical Laboratory Standards Institute CAP College of American Pathologists UK United Kingdom AUS Australia RCPA Royal College of Pathologists Australia QAP Quality Assurance Programs UK NEQAS United Kingdom National External Quality Assessment Scheme ICP-MS Inductively coupled plasma mass spectrometry
patients. Together, these were the first significant developments of what was to become the sweat test. Sweat Collection
The early techniques to induce sweat involved “thermal stress,” placing the patient in a heated room (908F/ 32.28C), for 1–2 hr.5 In 1955 a simpler method of thermal stress sweat collection was reported, with the patient wearing a plastic body bag tied loosely at the neck and covered in blankets for 60–90 min.6 This method was widely adopted although there were risks, including one reported fatality.6 Not surprisingly, this prompted a series of investigations into safer methods of sweat collection. Alternative strategies for detecting chloride in the skin through finger prints on plates impregnated with silver nitrate and potassium chromate was devised by Schwachman and Gahn.7 Similar methods were also reported by Webb, who placed filter paper soaked in silver nitrate between the fingers, and Gluck, who used a patch film test using the patient’s palm.8,9 Each of these tests relied on a color change when the silver nitrate reacted with the chloride in sweat. Although the silver nitrate tests were safer and simpler compared to the “thermal stress” tests, they were never fully validated and were rapidly replaced when the quantitative pilocarpine iontophoresis technique (QPIT) was developed. In 1959 Gibson and Cooke published the landmark method for sweat stimulation from eccrine sweat glands of the forearm, using topical application of pilocarpine through iontophoresis; the QPIT method.10 Pilocarpine is a parasympathomimetic alkaloid which acts on the cholinergic receptors by mimicking acetylcholine, a neurological transmitter. The charged pilocarpine is propelled transdermally by repulsive electromotive force using a small electrical current, that is, iontophoresis.11 This stimulates the muscarinic receptors on the eccrine sweat glands to induce sweat secretion.2,12 In the Gibson and Cooke QPIT method the sweat is collected onto gauze or filter paper. The collected sweat is then eluted from the collection material and analyzed using coulometry, colorimetry or ISE as a method for chloride and flame photometry for sodium. The Gibson and Cooke QPIT for sweat collection was not without its drawbacks. Electrode exposure, causing burns and blistering on the site of contact, were a known risk, although this was fundamentally due to technical errors from inexperienced sweat collectors exceeding the maximum electrical voltage or stimulation time.13,14 Over diagnosis of CF, due to high chloride results from poor sample collection as well as sample evaporation also Pediatric Pulmonology
108
Collie et al.
occurred.15,16 Despite these problems the Gibson and Cooke method remained the only recognized method of sweat collection for the next 25 years. In 1983 the Wescor (Logan, Utah) Macroduct system of sweat collection was introduced (See Fig. 1).17,18 This system restructured the conventional QPIT into a comparatively simplified collection method removing
the need for reagent preparation.19 The iontophoretic current passes through specially designed gel discs containing pilocarpine, which is delivered through the skin to the eccrine sweat glands by iontophoresis. The resulting sweat is collected into coiled capillary tubing. The sample is analyzed by a variety of techniques. The popularity of this system is largely attributed to its
Fig. 1. Gibson and Cooke and Wescor sweat collection consumables. A: Iontophroesis system with electrodes and straps (Wescor shown here). Wescor—B: Pilocarpine impregnated gel discs for sweat stimulation, C: Macroduct coil sweat collection devise, D: Macroduct collection tube. Gibson and Cooke—E: Gauze pad soaked in pilocarpine for sweat stimulation, F: Pre-weighed gauze pad for sweat collection, G: Pre-weighed storage pot containing collected sweat on gauze.
Pediatric Pulmonology
Advances in Cystic Fibrosis Sweat Chloride Testing
comparative automation of sweat stimulation and ease of use. The Wescor Macroduct was approved for sweat collection by the American Cystic Fibrosis Foundation (CFF) in 1990.18 Today, both the Gibson and Cooke QPIT and the Macroduct systems are recommended sweat collection methods for the investigation of CF and published comparison of these two systems report equivalent results.19–23 A significant difference between the Gibson and Cooke QPIT and Macroduct QPIT sweat collection systems relates to the sample matrix. The Gibson and Cooke QPIT sample is a diluted sample due to the need for elution from the collection padding. The Macroduct QPIT sample is collected directly into tubing and can be analyzed neat. The choice of collection technique therefore influences the method of analysis. Gibson and Cooke suggested reference intervals for sweat chloride (and sodium) which surprisingly, given the relative lack of rigour in collection of samples from healthy subjects, have stood the test of time. Further clinical contributions have been made through the development of reference intervals for most age groups.24,25 Through these developments, our understanding of CF has also changed with recognition that there is a spectrum of clinical manifestations relating to CFTR dysfunction and that the sweat test contributes to this understanding.26 THE SWEAT CHLORIDE TEST AS A MEASURE OF CFTR FUNCTION
The original observations of elevated sweat electrolytes in patients with CF had no recognized physiological basis
109
and seemed at odds with a disease that caused sticky mucous in the lungs, pancreatic ducts and gut. Observations from studies of the pancreatic ducts and nasal epithelium suggested the problem was one of electrolyte transport at epithelial surfaces. 27–29 However, it was analysis of the sweat ducts by Quinton30 that identified the basic defect to be specific to chloride ion transport. Knowledge of the electrolyte transport defect was fundamental in the efforts to discover the genetic basis of CF. In 1989, the CFTR protein and the common mutation associated with most cases of CF, was discovered on chromosome 7 (7q31.2).31–33 The structure of CFTR was confirmed as an epithelial electrolyte transport protein, predominantly for chloride but also regulating a separate sodium channel (ENaC). There are other chloride channels such as the outwardly rectifying chloride channel (ORCC) and the calcium activated chloride channel (CaCC) that are an alternative pathway for chloride transport. Normal CFTR function and the defect resulting from the common mutations are outlined in Figure 2. Following the original identification of the common CFTR mutation p.Phe508del (formerly DF508), other mutations were rapidly identified and there are now over 1,900 known mutations and polymorphisms.34 Nevertheless, most clinical laboratories test a limited number of the more common CFTR mutations. In addition CFTR sequencing is expensive and may yield sequence variations of unknown significance. For these reasons, genetic testing has not replaced the sweat chloride test as the major diagnostic tool for CF.35 This is reflected in the
Fig. 2. Diagram outlining the transportation of chloride and sodium through a normal, p. Phe508del and p.Gly551asp mutations of a sweat duct in the eccrine gland.
Pediatric Pulmonology
110
Collie et al.
consensus statements for the diagnosis of CF which rely on a combination of recognized clinical features, CFTR gene mutations and the QPIT sweat chloride test. 36,37 Investigations of the first drug to successfully restore CFTR function of the sweat gland recognized the value of the sweat test to monitor this function.38,39 In patients with CF, with the CFTR mutation p.Gly551asp (formally G551D), the mean sweat chloride level reduced from a mean of 100.4–52.5 mmol/L confirming the biological effect of the drug ivacaftor (Vertex Pharmaceuticals, Cambridge, MA) activating sweat gland CFTR function.39,40 The precise mechanism of this action remains unknown and there is speculation regarding whether there was any organ specific restoration of CFTR function, other than the sweat gland, but the sweat test played an important role in determining that the efficacy of ivacaftor was due to improved CFTR function in patients with the Gly551Asp mutation.39,41 As such, the future role of the sweat test is likely to include the monitoring of treatment success and compliance, in addition to its traditional role in the investigation of CF. STANDARDIZATION OF THE SWEAT CHLORIDE TEST: WHERE ARE WE TODAY?
Through the development of guidelines, there is one agreed methodology for sweat collection, pilocarpine iontophoresis, and several methodologies for sweat analysis. Some of these variations include which analyte is to be measured, how it is analyzed and what reference intervals are applicable to the results. Recent reports indicate that there is still a distinct lack of laboratory standardization in most countries, which
appears to be due to poor adherence to published guidelines. 42–44 Guidelines
The need for standardization of the collection, analysis and reporting of the sweat test was addressed by the first National Committee for Clinical Laboratory Standards (NCCLS—now known as the Clinical Laboratory Standards Institute or CLSI) guideline in 1994.45 Since then there has been refinement of sweat test methodologies, the development of guidelines in other countries and updates of the CLSI guideline. The following discussion covers the English language guidelines but acknowledges that other non-English procedures, such as the French Guideline, have been published.46 (A definition of standardization is provided in Table 1.47) Following the first version of the CLSI guideline for the sweat chloride test in 1994, CLSI published updated guidelines in 2000 and again in 2009.23,45,48 The CFF produced a guideline of their own in 2007 which was based on the 2nd CLSI guideline and the College of American Pathologists (CAP) Laboratory Accreditation Program Inspection Checklist.49 The United Kingdom (UK) developed an evidence based guideline for sweat chloride testing in 2003 as did Australia (AUS) in 2006.20,21 A comparison of these guidelines, highlighting critical aspects for standardization, can be seen in Table 2. Today, apart from CFF and CAP accreditation, there is still limited demonstrated enforcement of compliance with the guidelines. Without compliance, inconsistency in sweat chloride testing between laboratories is evident. For example, an Italian survey uncovered inconsistencies, including sweat
TABLE 1— Definitions Related to Methods of Analysis for Sweat Chloride Testing
Terminology
Description
Addition dilution
‘In relation to the Sweat Test the term “Addition Dilution” refers to the addition of a known concentration (e.g., Standard of Control) to a sample in order to calculate a result which would otherwise be below the sensitivity of the method’ Determines the concentration by measuring the absorbance of a specific wavelength of light. The intensity of the colour is directly proportional to concentration Analytical chemistry technique that utilizes an electrolysis reaction to measure the changes in resistance to the current between electrodes. The concentration of the titrant is equivalent to the current generated Converts the activity of a specific ion dissolved in a solution into an electrical potential which is measured by a voltmeter Inductively Coupled Plasma Mass Spectrometry or ICP-MS is an analytical technique used for elemental determinations. It was commercially introduced in 1983 and combines a high-temperature ICP source with a mass spectrometer. The plasma source converts the atoms of the elements in the sample to ions that are then separated and detected by the mass spectrometer Standardization as a concept is the agreement of test results. In relation to the sweat test, the term “standardization” incorporates all aspects of the test from patient eligibility through to clinical interpretation. For the analysis of sweat chloride, standardization specifically refers to the establishment of traceability to higher order reference materials and measurement procedures with the outcome of agreement with established trueness of the result
Colorimetry Coulometry Ion selective electrode ICP-MS
Standardization
Pediatric Pulmonology
5 min Max 30 min No information available within guideline
Sweat conductivity
Sodium—Flame photometry
Stimulation time Collection time Storage
Screening method
Method of analysis
Current
Chloride—Titration with chloride meter
0.075 g—Gauze/Filter paper 15 ml—Microbore tubing 0.075 g—Gauze/Filter paper 15 ml—Microbore tubing 4 mA
Minimum weight/ volume
Minimum age/weight of patient
Gauze or filter paper low in sodium and chloride OR Microbore tubing >48 hr/No minimum weight
1994
Medium of collection
Year
CLSI 1st Edition
Chloride—Titration with chloride meter. Advice against ISE. Other methods acceptable if validated
Sodium—Flame photometry
Sweat conductivity, Sweat Sodium (flame photometry)
5 min Max 30 min No information available within guideline
4 mA
0.075 g—Gauze/Filter paper 15 ml—Microbore tubing
Gauze or filter paper low in sodium and chloride OR Microbore tubing >48 hr/No minimum weight
2000
CLSI 2nd Edition
Chloride—Colorimetry, Coulometry, and ISE
Sweat conductivity— Wescor equipment only and a CV of 48 hr/No minimum weight
2007
CFF
Washed filter paper or gauze OR Wescor Macroduct collectors >2 weeks of age/>3 kg
2006
Australia
(Continued)
Other methods acceptable if validated
5 min Max 30 min Stable for 72 hr. Gauze or filter paper in a vial. Microcentrifuge tubes Sample must not be stored in Microbore tubing Sweat Conductivity (outside of cystic fibrosis accredited centres only) Chloride—Titration with chloridemeter. Caution using ISE
2.5–4 mA
Gauze or filter paper low in sodium and chloride OR Microbore tubing 2 weeks of age/>2 kg OR 48 hr of age if clinically important and sufficient sweat collected 0.075 g - Gauze/Filter paper 15 mL—Microbore tubing
2009
CLSI 3rd Edition
TABLE 2— Comparison of Evidence Based English Language Guidelines for the Standardization of Sweat Testing for Use in the Investigation of Cystic Fibrosis by the Australian Sweat Test Working Party (AUS), UK Multi-Disciplinary Working Group (UK), Cystic Fibrosis Foundation (CFF), CLSI and Clinical Laboratory Standards Institute (CLSI - formally NCCLS)
Advances in Cystic Fibrosis Sweat Chloride Testing 111
Pediatric Pulmonology
30 mmol/L considered abnormal 60 mmol/L supports CF diagnosis >60 mmol/L supports CF diagnosis
40–60 mmol/L equivocal
60 mmol/L supports CF diagnosis >60 mmol/L supports CF diagnosis Sweat Chloride Reference Intervals
2007 2006 2003 2000 1994 Year
CFF Australia United Kingdom CLSI 2nd Edition CLSI 1st Edition
TABLE 2— (Continued)
2009
Collie et al. CLSI 3rd Edition
112
Pediatric Pulmonology
collection times extending from
