Scandinavian Journal of Clinical & Laboratory Investigation, 2014; 74: 708–712
ORIGINAL ARTICLE
Lack of harmonization in sweat testing for cystic fibrosis – a national survey
ANNE LINDEGAARD CHRISTIANSEN & MADS NYBO Department of Clinical Biochemistry and Pharmacology, Odense University Hospital, Denmark Abstract Introduction. Sweat testing is used in the diagnosis of cystic fibrosis. Interpretation of the sweat test depends, however, on the method performed since conductivity, osmolality and chloride concentration all can be measured as part of a sweat test. The aim of this study was to investigate how performance of the test is organized in Denmark. Methods. Departments conducting the sweat test were contacted and interviewed following a premade questionnaire. They were asked about methods performed, applied NPU (Nomenclature for Properties and Units) code, reference interval, recommended interpretation and referred literature. Results. 14 departments performed the sweat test. One department measured chloride and sodium concentration, while 13 departments measured conductivity. One department used a non-existing NPU code, two departments applied NPU codes inconsistent with the method performed, four departments applied no NPU code and seven applied a correct NPU code. Ten of the departments measuring conductivity applied reference intervals. Nine departments measuring conductivity had recommendations of a normal area, a grey zone and a pathological value, while four departments only applied a normal and grey zone or a pathological value. Cut-off values for normal, grey and pathological areas were like the reference intervals inconsistent. Conclusion. There is inconsistent use of NPU codes, reference intervals and interpretation of sweat conductivity used in the process of diagnosing cystic fibrosis. Because diagnosing cystic fibrosis is a combined effort between local pediatric departments, biochemical and genetic departments and cystic fibrosis centers, a national harmonization is necessary to assure correct clinical use. Key Words: Chlorides, cystic fibrosis, diagnosis, screening and sweat
Introduction Cystic fibrosis (CF), caused by a mutation in the CF transmembrane conductance regulator (CFTR) gene, is the most common autosomal recessive disorder in a Caucasian population. The gene carrier frequency in Denmark is 1:30–35 with an incidence of 1:4760 [1]. In Denmark, there is no newborn screening for CF: Children with CF are diagnosed when symptoms appear or if there is a family history of CF. Sweat testing is of major importance in the process of diagnosing CF and is used along with phenotypic characteristics and gene mutation analysis. The indication for sweat testing is clinical suspicion, testing of siblings, and in some parts of the world it is also part of newborn screening programs [2,3]. When conducting the sweat test, sweating is stimulated locally by iontophoresis of pilocarpin into
the skin [4], where after sweat is collected on gauze, filter paper or in a Macroduct coil, Wescor®, for further analysis of conductivity, chloride concentration and/or osmolality [5–7]. Conductivity reflects the total amount of electrolytes, which results in a higher reference interval for conductivity compared to a direct chloride concentration measurement [5]. According to the American Cystic Fibrosis Foundation Consensus report and European Cystic Fibrosis Society only measurement of the chloride concentration in sweat can be used in the diagnosis of CF [2,8], while measurement of conductivity should be used as a preliminary analysis resulting in measurement of chloride concentration when conductivity is ⱖ 50 mmol/L [9]. However, there seems to be confusion regarding which methods are actually used and which reference intervals to apply within this area as indicated by
Correspondence: Anne Lindegaard Christiansen, Department of Clinical Biochemistry and Pharmacology, Odense University Hospital, Sdr. Boulevard 29, 5000 Odense C, Denmark. Tel: ⫹ 45 6541 3333. E-mail: [email protected] (Received 31 March 2014 ; accepted 8 August 2014 ) ISSN 0036-5513 print/ISSN 1502-7686 online © 2014 Informa Healthcare DOI: 10.3109/00365513.2014.953992
Lack of harmonization in sweat testing previous surveys [10–13]. Of note, there are, to our knowledge, no published Danish guidelines for performing the sweat test or for diagnosing CF. The aim of this study was therefore to elucidate methods, NPU codes (Nomenclature for Properties and Units), reference intervals and recommended interpretations used for sweat tests performed at Danish hospitals.
and 2 (14%) at a Department of Clinical Physiology. In two instances (14%) the sweat test was performed at a pediatric ward and conducted by a nurse (Table II). Three systems were used for the sweat test: The Macroduct system, the NanoductÒ, WescorÒ (both measuring the sweat conductivity) and a chloride concentration measurement: •
Methods All major departments of Clinical Biochemistry in the regions of Denmark were contacted by telephone to elucidate if they, any of the satellite Clinical Biochemistry departments or other clinical departments in their region performed the sweat test. All departments were asked from a premade questionnaire containing five questions with focus on the analysis method, NPU codes, the reference interval, recommended interpretation and referred literature. All information available (e.g. lab information from the Internet, validation reports, standard operating procedures) entered the survey. The study was performed between January and December 2013. In general, NPU codes are an international terminology used in laboratory medicine [14]. NPU code represents a specific analysis, and when applying an NPU code information of a specific component measured in a specific system is identified along with the property of the component measured and the unit by which the property is measured. In Denmark, NPU codes are used at all Departments of Biochemistry in order to assure comparability of results (Table I).
Results A total of 14 departments conducted the sweat test in Denmark and all departments were willing to answer the questionnaire. Of these 14 departments, the sweat test was performed at a laboratory by a laboratory technician in 12 departments (86%), of which 10 (71%) was at a Department of Biochemistry
709
•
•
The Macroduct system consists of the Webster Sweat Inducer, the Macroduct Sweat Collector and the Sweat-Check Analyzer. The NanoductÒ is an integrated system for the induction of iontophoresis and sweat analysis. The NanoductÒ system is less time-consuming and requires a smaller volume than the Macroduct system. Chloride and sodium concentrations were measured using an indirect ion-selective electrode on Modular analytics system (Roche Diagnostics, Switzerland) after sweat stimulation using the Webster Sweat inducer and Macroduct Sweat Collector.
One department measured chloride and sodium concentrations, while all other departments measured conductivity. Five departments used the Macroduct system, while the remaining eight used NanoductÒ. NPU codes were only used by the Departments of Biochemistry, but despite performing the same test there was discordant use of NPU codes for the laboratories conducting conductivity (Table II): Overall, six of the 13 departments performing conductivity used the NPU code 21552, while one department used a non-existing NPU code 08755 and two other departments used NPU codes not applicable for measurement of conductivity (Tables I and II). Reference intervals were referred by 11 of the departments: Seven of the departments performing the conductivity test had a reference interval ⬍ 60 mmol/L, but it varied considerably (⬍ 60, 3–60 or 17–60) and were given in either mmol/L or arbitrary units (AU). Two other departments had ⬍ 65 mmol/L,
Table I. Nomenclature for properties and units (NPU) codes. Definition of NPU codes used for sweat testing. NPU
Definition
01537 03430 08755 17183 18285 18328 18331 18335 18338 21552
Sweat (spec.) – chloride; subst.c. ⫽ ? mmol/L Sweat (spec.) – sodium ion; subst.c. ⫽ ? mmol/L Does not exist Skin surface (spec.) – sweat tolerance; k-o-p (list; Pilocarpine i.c.; proc.) Sweat (spec.) – sweat; vol. (proc.) ⫽ ? μL Sweat (Arm; right) – chloride; subst.c. ⫽ ? mmol/L Sweat (Arm; right) – sodium ion; subst.c. ⫽ ? mmol/L Sweat (Arm; left) – chloride; subst.c. ⫽ ? mmol/L Sweat (Arm; left) – sodium ion; subst.c. ⫽ ? mmol/L Sweat (spec.) – sweat; arb.conductivity (proc.) ⫽ ? (p.d.u.)
Arb., arbitrary; k-o-p, kind-of-property; proc, procedure; p.d.u, procedure defined unit; spec, specification; subst.c, substance concentration; vol, volume.
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Table II. Results of the survey. List of departments in Denmark conducting the sweat test. Department
NPU code
Method
Reference interval
1 2
Dept. of Biochemistry Dept. of Biochemistry
Nanoduct® Nanoduct®
21552 21552
⬍ 60 mmol/L eq NaCl
3 4 5 6
Paediatric Dept. Dept. of Biochemistry Dept. of Biochemistry Dept. of Biochemistry
NanoductÒ NanoductÒ NanoductÒ NanoductÒ
None 21552 21552 21552
⬍ 16 years: 17–60 AU 0–16 years: 17–60 AU 0–200 years: ⬍ 60 AU
7 8
Paediatric Dept. Dept. of Biochemistry
NanoductÒ NanoductÒ
None 01537
⬍ 60 0–16 years: 3–60 mmol/L
9
Dept. of Biochemistry
The Macroduct system
21552
0–16 years: 17–60 AU
10
Dept. of Biochemistry
The Macroduct system
⬍ 65 mmol/L
11 12 13
Dept. of Clinical Physiology Dept. of Clinical Physiology Dept. of Biochemistry
The Macroduct system The Macroduct system The Macroduct system
17183 03430 18285 None None 08755
14
Dept. of Biochemistry
Chloride and sodium concentration
17183 18328 18331 18335 18338
Sweat – chloride: ⬍ 30 mmol/L Sweat – sodium: ⬍ 40 mmol/L
Literature [15] NanoductÒ, Wescor, 2001/2006 [15] [17] NanoductÒ, Wescor [15] [15] [5] [15] NanoductÒ, Wescor NanoductÒ, Wescor, 2001/2004 [18] Macroduct system, Wescor [5] Macroduct system, Wescor [16] [16]
⬍ 65 mmol/L 18–200 years: 13–87 mmol/L
[5] [4] [19] [20] [21] [22]
Eq, equivalent; AU, arbitrary units.
while one department had 13–87 mmol/L as reference interval. The department measuring chloride and sodium concentrations had a reference interval of ⬍ 30 mmol/L and ⬍ 40 mmol/L, respectively. Six of the departments applied age to the reference interval: Four of them applied 0–16 years, one department used 18–200 years, while another department applied a reference interval spanning from 0–200 years (Table II). As shown in Figure 1, different interpretations were recommended at the departments performing
Figure 1. Recommended interpretation of the sweat test. Reference intervals and recommended interpretation for sweat conductivity in Denmark. Numbers indicate the different departments as listed in Table II. AU, arbitrary units. , reference interval and/or normal area; , grey zone; , pathological value
sweat conductivity, despite performing the same method. Majority of the departments referred to values ⬍ 60/61 AU or mmol/L as normal or indicating that a CF diagnosis was unlikely. Values between 60/61–80 AU or mmol/L were in a ‘grey zone’ with recommendation of repeating the test or conducting further analysis. Values above 80 AU or mmol/L were referred to as either pathologic or consistent with or supportive of CF. Two departments had the same recommendations, but with different cut-off values (⬍ 65, 65–90 and ⬎ 90 mmol/L, while the other used ⬍ 60, 60–90 and ⬎ 90 mmol/L). One department recommended that ⬎ 60 mmol/L required further investigations. The department with a reference interval of 13–87 mmol/L stated that an increased value indicated CF. One department had a recommendation that values between 45–65 mmol/L were in a grey zone and the test should be repeated, and also applying a reference interval ⬍ 65 mmol/L. Finally, one of the pediatric departments recommended ⬍ 60 as normal and 60–81 as an uncertain result without applying any units. The department measuring chloride concentration did not recommend any interpretation because the clinicians interpreted the results themselves (Table II and Figure 1). All departments except one could refer to literature from where recommended interpretation was
Lack of harmonization in sweat testing adapted. Overall, departments using NanoductÒ referred to Desax et al. [15] and/or the manual from WescorÒ, while departments using the Macroduct system referred to Hammond et al. [5], Baumer [16] and/or the Manual, WescorÒ (Table II).
Discussion In Denmark, one department measures chloride concentration and 13 departments measure conductivity when sweat test is requested. Despite this relatively simple arrangement there are an unfortunate discordant use of reference intervals and NPU codes and no consensus regarding recommended interpretations. We will here try to elucidate the cause of this and describe the impact it can have on a diagnostic approach. There are different approaches to the CF diagnosis, e.g. clinical symptoms combined with the finding of a known CF mutation in the CFTR genes or a positive sweat test. The result and the interpretation of the sweat test are highly dependent on the preanalytical, analytical and postanalytical quality. Several international guidelines for performing the sweat test have therefore been published. According to the European and American guidelines only measurement of chloride concentration in sweat can be used as diagnostic tool, not conductivity [2,8,9]. However, only one department in Denmark performed the sweat test measuring chloride concentrations. To our knowledge the diagnostic function is not centralized in Denmark, and the fact that only one department performs the diagnostic test correctly could indicate that the diagnosis is either based on the wrong method (according to the mentioned guidelines) or that the sweat test is followed by genetic testing (which was not part of our investigation). Currently, there is an initiative from the Danish Cystic Fibrosis Association trying to establish a National screening program, and based on the findings from this study we will encourage the Danish Society of Clinical Biochemistry to advocate for a harmonized screening program. Three departments used NPU codes inconsistent with the actual method performed (Table I and II). The correct NPU code for sweat conductivity is NPU 21552 and it was not used by all the departments performing the conductivity test. NPU 01537 and 03430 used by departments measuring conductivity can be applied on chloride and sodium concentration measurements in sweat, respectively, with no specification of located collection site. Of note, one department used a non-existing NPU code. The fact that NPU codes for measuring concentrations are used for conductivity tests reflects the fact that the sweat test in Denmark is an orphan test. When the laboratories are not completely in charge of the methods performed the clinicians could receive
711
wrong information and interpret the results erroneously. It is therefore of the utmost importance that especially esoteric analyses are handled with proper laboratory practice in order to assure a high quality and a careful interpretation. A way to circumvent this could be to perform the sweat test at only a few laboratories or perhaps just one in such a small country as Denmark. Concerning the interpretation most departments had ⬍ 60 mmol/L as normal, a grey zone area between 60/61–80 mmol/L and a pathological value ⬎ 80 mmol/L. Departments recommending these interpretations and using the NanoductÒ mostly referred to Desax et al. [15] and/or the NanoductÒ manual. Of note, the department using the non-existing NPU code had (as the only department) a reference interval of 13–87 mmol/L for the age group 18–200 years. This reference interval is the sweat conductivity range from 471 non-CF patients with an age range of 3 days to 78 years and a median of 28 weeks [5]. The two departments that recommended ⬎ 90 mmol/L as a pathological value referred to Baumer [16]; however, one of the departments had ⬍ 65 mmol/L as normal/CF unlikely, while Baumer recommend ⬍ 60 mmol/L as CF unlikely and ⬎ 90 mmol/L as supportive for a diagnosis of CF. Only one department had a grey zone of 45–65 mmol/L and did as the only department not refer to any literature or manual. Of note, no department applied a reference interval or interpretation applicable for the chloride concentration in sweat, which according to the American Cystic Fibrosis Foundation is ⬍ 40 mmol/L ⫽ negative, 40–60 ⫽ borderline/indeterminate and ⬎ 60 ⫽ consistent with the diagnosis of CF [6]. Overall, there were differences in the applied reference intervals and recommended interpretations, and the recommended interpretations were based on different literature. Altogether, a very inhomogeneous picture which calls for harmonization. Sweat testing is used in different diagnostic constellations around the world as the indication for sweat testing can be based on clinical suspicion, a family history or as part of a newborn screening program, respectively. Applying a test under different circumstances changes the predictive values because of the different prevalence in the different clinical settings, and it is therefore important that clinicians interpreting the results take this into account. In Denmark, the indication for sweat testing is clinical suspicion or family history as there is not yet a national CF screening program for newborns (as in many other European countries) [8]. If such a program was to be initiated the way sweat testing is used would change and the number of sweat tests performed would be expected to increase. Therefore, it is important that there is a national agreement on the methods used, reference intervals applied and last, but not least the interpretation of the results. Since the sweat test is performed by nurses at
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paediatric wards as well as technicians at laboratory departments a straight forward way of reporting the results is also important. The lack of harmonization found in this study could very well impact the diagnosis and treatment of the patients: Clinicians may think that a conductivity test is confirmatory for CF (which as afore mentioned is not recommended by international guidelines). Also, applying a NPU code for chloride concentration instead of for sweat conductivity may confuse interpretation of the result and perhaps lead to further unnecessary investigations and concern for the patient’s parents, because the reference intervals for chloride concentration and conductivity are different. As for any security system, correct use ensures harmonization, understanding and transparency, while incorrect use endangers the same values. If a certain system is used, it often replaces other safety rules, i.e. ‘old ways’ of assuring correctness, traceability etc. It is therefore of the utmost importance that everybody involved adheres to rules of the system in use, e.g. the NPU codes. In conclusion, the performance and interpretation of the sweat test (as a good example of an esoteric analysis) is not harmonized, which will influence the diagnostic use of this test. In general, it is important to upgrade the cooperation on this area, especially between different medical specialties since, e.g. the diagnosis of CF is a combined effort of local paediatric departments, biochemical departments and CF centres. The suggested national organization of the sweat test should also be considered for other highly specialized analyses to avoid this ‘orphan test phenomenon’.
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
Declaration of interest: The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper. References [1] Nielsen OH, Thomsen BL, Green A, Andersen PK, Hauge M, Schiotz PO. Cystic fibrosis in Denmark 1945 to 1985. An analysis of incidence, mortality and influence of centralized treatment on survival. Acta Paediatr Scand 1988;77:836–41. [2] Farrell PM, Rosenstein BJ, White TB, Accurso FJ, Castellani C, Cutting GR, Durie PR, LeGrys VA, Massie J, Parad RB, Rock MJ, Campbell PW, III. Guidelines for diagnosis of cystic fibrosis in newborns through older adults: Cystic Fibrosis Foundation consensus report. J Pediatr 2008;153:S4–14. [3] Boeck KD, Wilschanski M, Castellani C, Taylor C, Cuppens H, Dodge J, Sinaasappel M. Cystic fibrosis: terminology and diagnostic algorithms. Thorax 2006;61: 627–35. [4] Gibson LE, Cooke RE. A test for concentration of electrolytes in sweat in cystic fibrosis of the pancreas utilizing pilocarpine by iontophoresis. Pediatrics 1959;23:545–9. [5] Hammond KB, Turcios NL, Gibson LE. Clinical evaluation of the macroduct sweat collection system and conductivity
[16]
[17]
[18] [19]
[20]
[21]
[22]
analyzer in the diagnosis of cystic fibrosis. J Pediatr 1994;124:255–60. LeGrys VA, Yankaskas JR, Quittell LM, Marshall BC, Mogayzel PJ Jr. Diagnostic sweat testing: The Cystic Fibrosis Foundation Guidelines. J Pediatr 2007;151: 85–9. Schoni MH, Kraemer R, Bahler P, Rossi E. Early diagnosis of cystic fibrosis by means of sweat microosmometry. J Pediatr 1984;104:691–4. Castellani C, Southern KW, Brownlee K, Roelse JD, Duff A, Farrell M, Mehta A, Munck A, Pollitt R, Sermet-Gaudelus I, Wilcken B, Ballmann M, Corbetta C, de Monsterol II, Farrell P, Feilcke M, Ferec C, Gartner S, Gaskin K, Hammermann J, Kashirskaya N, Loeber G, Macek M Jr, Mehta G, Reiman A, Rizzotti P, Sammon A, Sands D, Smyth A, Sommerburg O, Torresani T, Travert G, Vernooij A, Elborn S. European best practice guidelines for cystic fibrosis neonatal screening. J Cyst Fibros 2009;8: 153–73. The Clinical and Laboratory Standards Institute (CLSI). Sweat testing: sample collection and quantitative chloride analysis; Approved guideline – 3rd ed. C34-A3; 2009. Coakley J, Scott S, Mackay R, Greaves R, Jolly L, Massie J, Mishra A, Bransden A, Doery JC, Chiriano A, Robins H. Sweat testing for cystic fibrosis: standards of performance in Australasia. Ann Clin Biochem 2009;46:332–7. Barben J, Casaulta C, Spinas R, Schoni MH. Sweat testing practice in Swiss hospitals. Swiss Med Wkly 2007;137: 192–8. Mackay R, George P, Kirk J. Sweat testing for cystic fibrosis: a review of New Zealand laboratories. J Paediatr Child Health 2006;42:160–4. LeGrys VA. Assesment of sweat-testing practices for the diagnose of cystic fibrosis. Arch Pathol Lab Med 2001; 125:1420–4. Pontet F, Magdal PU, Fuentes-Arderiu X, Nordin G, Bruunshuus I, Ihalainen J, Karlsson D, Forsum U, Dybkaer R, Schadow G, Kuelpmann W, Ferard G, Kang D, McDonald C, Hill G. Clinical laboratory sciences data transmission: the NPU coding system. Stud Health Technol Inform 2009; 150:265–9. Desax MC, Ammann RA, Hammer J, Schoeni MH, Barben J. Nanoduct sweat testing for rapid diagnosis in newborns, infants and children with cystic fibrosis. Eur J Pediatr 2008;167:299–304. Baumer JH. Evidence based guidelines for the performance of the sweat test for the investigation of cystic fibrosis in the UK. Arch Dis Child 2003;88:1126–7. Barben J, Ammann RA, Metlagel A, Schoeni MH. Conductivity determined by a new sweat analyzer compared with chloride concentrations for the diagnosis of cystic fibrosis. J Pediatr 2005;146:183–8. Lyngbye J. Laboratoriemedicin. 2nd ed. Nyt Nordisk Forlag Arnold Busk; 2010 [in Danish]. Mastella G, Di CG, Borruso A, Menin L, Zanolla L. Reliability of sweat-testing by the Macroduct collection method combined with conductivity analysis in comparison with the classic Gibson and Cooke technique. Acta Paediatr 2000;89:933–7. Green A, Kirk J. Guidelines for the performance of the sweat test for the diagnosis of cystic fibrosis. Ann Clin Biochem 2007;44:25–34. The Clinical and Laboratory Standards Institute (CLSI). Sweat testing: sample collection and quantitative chloride analysis; Approved guideline – 2nd ed. C34-A2; 2000. Guidelines development group. Guidelines for the Performance of the Sweat test for the Investigation of Cystic Fibrosis in the UK. 2003 November. Available at: http:// www.acb.org.uk/docs/default-source/guidelines/Sweat. pdf?sfvrsn ⫽ 0 [accessed 31 March 2014].
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ORIGINAL ARTICLE
Lack of harmonization in sweat testing for cystic fibrosis – a national survey
ANNE LINDEGAARD CHRISTIANSEN & MADS NYBO Department of Clinical Biochemistry and Pharmacology, Odense University Hospital, Denmark Abstract Introduction. Sweat testing is used in the diagnosis of cystic fibrosis. Interpretation of the sweat test depends, however, on the method performed since conductivity, osmolality and chloride concentration all can be measured as part of a sweat test. The aim of this study was to investigate how performance of the test is organized in Denmark. Methods. Departments conducting the sweat test were contacted and interviewed following a premade questionnaire. They were asked about methods performed, applied NPU (Nomenclature for Properties and Units) code, reference interval, recommended interpretation and referred literature. Results. 14 departments performed the sweat test. One department measured chloride and sodium concentration, while 13 departments measured conductivity. One department used a non-existing NPU code, two departments applied NPU codes inconsistent with the method performed, four departments applied no NPU code and seven applied a correct NPU code. Ten of the departments measuring conductivity applied reference intervals. Nine departments measuring conductivity had recommendations of a normal area, a grey zone and a pathological value, while four departments only applied a normal and grey zone or a pathological value. Cut-off values for normal, grey and pathological areas were like the reference intervals inconsistent. Conclusion. There is inconsistent use of NPU codes, reference intervals and interpretation of sweat conductivity used in the process of diagnosing cystic fibrosis. Because diagnosing cystic fibrosis is a combined effort between local pediatric departments, biochemical and genetic departments and cystic fibrosis centers, a national harmonization is necessary to assure correct clinical use. Key Words: Chlorides, cystic fibrosis, diagnosis, screening and sweat
Introduction Cystic fibrosis (CF), caused by a mutation in the CF transmembrane conductance regulator (CFTR) gene, is the most common autosomal recessive disorder in a Caucasian population. The gene carrier frequency in Denmark is 1:30–35 with an incidence of 1:4760 [1]. In Denmark, there is no newborn screening for CF: Children with CF are diagnosed when symptoms appear or if there is a family history of CF. Sweat testing is of major importance in the process of diagnosing CF and is used along with phenotypic characteristics and gene mutation analysis. The indication for sweat testing is clinical suspicion, testing of siblings, and in some parts of the world it is also part of newborn screening programs [2,3]. When conducting the sweat test, sweating is stimulated locally by iontophoresis of pilocarpin into
the skin [4], where after sweat is collected on gauze, filter paper or in a Macroduct coil, Wescor®, for further analysis of conductivity, chloride concentration and/or osmolality [5–7]. Conductivity reflects the total amount of electrolytes, which results in a higher reference interval for conductivity compared to a direct chloride concentration measurement [5]. According to the American Cystic Fibrosis Foundation Consensus report and European Cystic Fibrosis Society only measurement of the chloride concentration in sweat can be used in the diagnosis of CF [2,8], while measurement of conductivity should be used as a preliminary analysis resulting in measurement of chloride concentration when conductivity is ⱖ 50 mmol/L [9]. However, there seems to be confusion regarding which methods are actually used and which reference intervals to apply within this area as indicated by
Correspondence: Anne Lindegaard Christiansen, Department of Clinical Biochemistry and Pharmacology, Odense University Hospital, Sdr. Boulevard 29, 5000 Odense C, Denmark. Tel: ⫹ 45 6541 3333. E-mail: [email protected] (Received 31 March 2014 ; accepted 8 August 2014 ) ISSN 0036-5513 print/ISSN 1502-7686 online © 2014 Informa Healthcare DOI: 10.3109/00365513.2014.953992
Lack of harmonization in sweat testing previous surveys [10–13]. Of note, there are, to our knowledge, no published Danish guidelines for performing the sweat test or for diagnosing CF. The aim of this study was therefore to elucidate methods, NPU codes (Nomenclature for Properties and Units), reference intervals and recommended interpretations used for sweat tests performed at Danish hospitals.
and 2 (14%) at a Department of Clinical Physiology. In two instances (14%) the sweat test was performed at a pediatric ward and conducted by a nurse (Table II). Three systems were used for the sweat test: The Macroduct system, the NanoductÒ, WescorÒ (both measuring the sweat conductivity) and a chloride concentration measurement: •
Methods All major departments of Clinical Biochemistry in the regions of Denmark were contacted by telephone to elucidate if they, any of the satellite Clinical Biochemistry departments or other clinical departments in their region performed the sweat test. All departments were asked from a premade questionnaire containing five questions with focus on the analysis method, NPU codes, the reference interval, recommended interpretation and referred literature. All information available (e.g. lab information from the Internet, validation reports, standard operating procedures) entered the survey. The study was performed between January and December 2013. In general, NPU codes are an international terminology used in laboratory medicine [14]. NPU code represents a specific analysis, and when applying an NPU code information of a specific component measured in a specific system is identified along with the property of the component measured and the unit by which the property is measured. In Denmark, NPU codes are used at all Departments of Biochemistry in order to assure comparability of results (Table I).
Results A total of 14 departments conducted the sweat test in Denmark and all departments were willing to answer the questionnaire. Of these 14 departments, the sweat test was performed at a laboratory by a laboratory technician in 12 departments (86%), of which 10 (71%) was at a Department of Biochemistry
709
•
•
The Macroduct system consists of the Webster Sweat Inducer, the Macroduct Sweat Collector and the Sweat-Check Analyzer. The NanoductÒ is an integrated system for the induction of iontophoresis and sweat analysis. The NanoductÒ system is less time-consuming and requires a smaller volume than the Macroduct system. Chloride and sodium concentrations were measured using an indirect ion-selective electrode on Modular analytics system (Roche Diagnostics, Switzerland) after sweat stimulation using the Webster Sweat inducer and Macroduct Sweat Collector.
One department measured chloride and sodium concentrations, while all other departments measured conductivity. Five departments used the Macroduct system, while the remaining eight used NanoductÒ. NPU codes were only used by the Departments of Biochemistry, but despite performing the same test there was discordant use of NPU codes for the laboratories conducting conductivity (Table II): Overall, six of the 13 departments performing conductivity used the NPU code 21552, while one department used a non-existing NPU code 08755 and two other departments used NPU codes not applicable for measurement of conductivity (Tables I and II). Reference intervals were referred by 11 of the departments: Seven of the departments performing the conductivity test had a reference interval ⬍ 60 mmol/L, but it varied considerably (⬍ 60, 3–60 or 17–60) and were given in either mmol/L or arbitrary units (AU). Two other departments had ⬍ 65 mmol/L,
Table I. Nomenclature for properties and units (NPU) codes. Definition of NPU codes used for sweat testing. NPU
Definition
01537 03430 08755 17183 18285 18328 18331 18335 18338 21552
Sweat (spec.) – chloride; subst.c. ⫽ ? mmol/L Sweat (spec.) – sodium ion; subst.c. ⫽ ? mmol/L Does not exist Skin surface (spec.) – sweat tolerance; k-o-p (list; Pilocarpine i.c.; proc.) Sweat (spec.) – sweat; vol. (proc.) ⫽ ? μL Sweat (Arm; right) – chloride; subst.c. ⫽ ? mmol/L Sweat (Arm; right) – sodium ion; subst.c. ⫽ ? mmol/L Sweat (Arm; left) – chloride; subst.c. ⫽ ? mmol/L Sweat (Arm; left) – sodium ion; subst.c. ⫽ ? mmol/L Sweat (spec.) – sweat; arb.conductivity (proc.) ⫽ ? (p.d.u.)
Arb., arbitrary; k-o-p, kind-of-property; proc, procedure; p.d.u, procedure defined unit; spec, specification; subst.c, substance concentration; vol, volume.
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Table II. Results of the survey. List of departments in Denmark conducting the sweat test. Department
NPU code
Method
Reference interval
1 2
Dept. of Biochemistry Dept. of Biochemistry
Nanoduct® Nanoduct®
21552 21552
⬍ 60 mmol/L eq NaCl
3 4 5 6
Paediatric Dept. Dept. of Biochemistry Dept. of Biochemistry Dept. of Biochemistry
NanoductÒ NanoductÒ NanoductÒ NanoductÒ
None 21552 21552 21552
⬍ 16 years: 17–60 AU 0–16 years: 17–60 AU 0–200 years: ⬍ 60 AU
7 8
Paediatric Dept. Dept. of Biochemistry
NanoductÒ NanoductÒ
None 01537
⬍ 60 0–16 years: 3–60 mmol/L
9
Dept. of Biochemistry
The Macroduct system
21552
0–16 years: 17–60 AU
10
Dept. of Biochemistry
The Macroduct system
⬍ 65 mmol/L
11 12 13
Dept. of Clinical Physiology Dept. of Clinical Physiology Dept. of Biochemistry
The Macroduct system The Macroduct system The Macroduct system
17183 03430 18285 None None 08755
14
Dept. of Biochemistry
Chloride and sodium concentration
17183 18328 18331 18335 18338
Sweat – chloride: ⬍ 30 mmol/L Sweat – sodium: ⬍ 40 mmol/L
Literature [15] NanoductÒ, Wescor, 2001/2006 [15] [17] NanoductÒ, Wescor [15] [15] [5] [15] NanoductÒ, Wescor NanoductÒ, Wescor, 2001/2004 [18] Macroduct system, Wescor [5] Macroduct system, Wescor [16] [16]
⬍ 65 mmol/L 18–200 years: 13–87 mmol/L
[5] [4] [19] [20] [21] [22]
Eq, equivalent; AU, arbitrary units.
while one department had 13–87 mmol/L as reference interval. The department measuring chloride and sodium concentrations had a reference interval of ⬍ 30 mmol/L and ⬍ 40 mmol/L, respectively. Six of the departments applied age to the reference interval: Four of them applied 0–16 years, one department used 18–200 years, while another department applied a reference interval spanning from 0–200 years (Table II). As shown in Figure 1, different interpretations were recommended at the departments performing
Figure 1. Recommended interpretation of the sweat test. Reference intervals and recommended interpretation for sweat conductivity in Denmark. Numbers indicate the different departments as listed in Table II. AU, arbitrary units. , reference interval and/or normal area; , grey zone; , pathological value
sweat conductivity, despite performing the same method. Majority of the departments referred to values ⬍ 60/61 AU or mmol/L as normal or indicating that a CF diagnosis was unlikely. Values between 60/61–80 AU or mmol/L were in a ‘grey zone’ with recommendation of repeating the test or conducting further analysis. Values above 80 AU or mmol/L were referred to as either pathologic or consistent with or supportive of CF. Two departments had the same recommendations, but with different cut-off values (⬍ 65, 65–90 and ⬎ 90 mmol/L, while the other used ⬍ 60, 60–90 and ⬎ 90 mmol/L). One department recommended that ⬎ 60 mmol/L required further investigations. The department with a reference interval of 13–87 mmol/L stated that an increased value indicated CF. One department had a recommendation that values between 45–65 mmol/L were in a grey zone and the test should be repeated, and also applying a reference interval ⬍ 65 mmol/L. Finally, one of the pediatric departments recommended ⬍ 60 as normal and 60–81 as an uncertain result without applying any units. The department measuring chloride concentration did not recommend any interpretation because the clinicians interpreted the results themselves (Table II and Figure 1). All departments except one could refer to literature from where recommended interpretation was
Lack of harmonization in sweat testing adapted. Overall, departments using NanoductÒ referred to Desax et al. [15] and/or the manual from WescorÒ, while departments using the Macroduct system referred to Hammond et al. [5], Baumer [16] and/or the Manual, WescorÒ (Table II).
Discussion In Denmark, one department measures chloride concentration and 13 departments measure conductivity when sweat test is requested. Despite this relatively simple arrangement there are an unfortunate discordant use of reference intervals and NPU codes and no consensus regarding recommended interpretations. We will here try to elucidate the cause of this and describe the impact it can have on a diagnostic approach. There are different approaches to the CF diagnosis, e.g. clinical symptoms combined with the finding of a known CF mutation in the CFTR genes or a positive sweat test. The result and the interpretation of the sweat test are highly dependent on the preanalytical, analytical and postanalytical quality. Several international guidelines for performing the sweat test have therefore been published. According to the European and American guidelines only measurement of chloride concentration in sweat can be used as diagnostic tool, not conductivity [2,8,9]. However, only one department in Denmark performed the sweat test measuring chloride concentrations. To our knowledge the diagnostic function is not centralized in Denmark, and the fact that only one department performs the diagnostic test correctly could indicate that the diagnosis is either based on the wrong method (according to the mentioned guidelines) or that the sweat test is followed by genetic testing (which was not part of our investigation). Currently, there is an initiative from the Danish Cystic Fibrosis Association trying to establish a National screening program, and based on the findings from this study we will encourage the Danish Society of Clinical Biochemistry to advocate for a harmonized screening program. Three departments used NPU codes inconsistent with the actual method performed (Table I and II). The correct NPU code for sweat conductivity is NPU 21552 and it was not used by all the departments performing the conductivity test. NPU 01537 and 03430 used by departments measuring conductivity can be applied on chloride and sodium concentration measurements in sweat, respectively, with no specification of located collection site. Of note, one department used a non-existing NPU code. The fact that NPU codes for measuring concentrations are used for conductivity tests reflects the fact that the sweat test in Denmark is an orphan test. When the laboratories are not completely in charge of the methods performed the clinicians could receive
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wrong information and interpret the results erroneously. It is therefore of the utmost importance that especially esoteric analyses are handled with proper laboratory practice in order to assure a high quality and a careful interpretation. A way to circumvent this could be to perform the sweat test at only a few laboratories or perhaps just one in such a small country as Denmark. Concerning the interpretation most departments had ⬍ 60 mmol/L as normal, a grey zone area between 60/61–80 mmol/L and a pathological value ⬎ 80 mmol/L. Departments recommending these interpretations and using the NanoductÒ mostly referred to Desax et al. [15] and/or the NanoductÒ manual. Of note, the department using the non-existing NPU code had (as the only department) a reference interval of 13–87 mmol/L for the age group 18–200 years. This reference interval is the sweat conductivity range from 471 non-CF patients with an age range of 3 days to 78 years and a median of 28 weeks [5]. The two departments that recommended ⬎ 90 mmol/L as a pathological value referred to Baumer [16]; however, one of the departments had ⬍ 65 mmol/L as normal/CF unlikely, while Baumer recommend ⬍ 60 mmol/L as CF unlikely and ⬎ 90 mmol/L as supportive for a diagnosis of CF. Only one department had a grey zone of 45–65 mmol/L and did as the only department not refer to any literature or manual. Of note, no department applied a reference interval or interpretation applicable for the chloride concentration in sweat, which according to the American Cystic Fibrosis Foundation is ⬍ 40 mmol/L ⫽ negative, 40–60 ⫽ borderline/indeterminate and ⬎ 60 ⫽ consistent with the diagnosis of CF [6]. Overall, there were differences in the applied reference intervals and recommended interpretations, and the recommended interpretations were based on different literature. Altogether, a very inhomogeneous picture which calls for harmonization. Sweat testing is used in different diagnostic constellations around the world as the indication for sweat testing can be based on clinical suspicion, a family history or as part of a newborn screening program, respectively. Applying a test under different circumstances changes the predictive values because of the different prevalence in the different clinical settings, and it is therefore important that clinicians interpreting the results take this into account. In Denmark, the indication for sweat testing is clinical suspicion or family history as there is not yet a national CF screening program for newborns (as in many other European countries) [8]. If such a program was to be initiated the way sweat testing is used would change and the number of sweat tests performed would be expected to increase. Therefore, it is important that there is a national agreement on the methods used, reference intervals applied and last, but not least the interpretation of the results. Since the sweat test is performed by nurses at
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paediatric wards as well as technicians at laboratory departments a straight forward way of reporting the results is also important. The lack of harmonization found in this study could very well impact the diagnosis and treatment of the patients: Clinicians may think that a conductivity test is confirmatory for CF (which as afore mentioned is not recommended by international guidelines). Also, applying a NPU code for chloride concentration instead of for sweat conductivity may confuse interpretation of the result and perhaps lead to further unnecessary investigations and concern for the patient’s parents, because the reference intervals for chloride concentration and conductivity are different. As for any security system, correct use ensures harmonization, understanding and transparency, while incorrect use endangers the same values. If a certain system is used, it often replaces other safety rules, i.e. ‘old ways’ of assuring correctness, traceability etc. It is therefore of the utmost importance that everybody involved adheres to rules of the system in use, e.g. the NPU codes. In conclusion, the performance and interpretation of the sweat test (as a good example of an esoteric analysis) is not harmonized, which will influence the diagnostic use of this test. In general, it is important to upgrade the cooperation on this area, especially between different medical specialties since, e.g. the diagnosis of CF is a combined effort of local paediatric departments, biochemical departments and CF centres. The suggested national organization of the sweat test should also be considered for other highly specialized analyses to avoid this ‘orphan test phenomenon’.
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
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