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31.van Royen N, Piek JJ, Legemate DA, Schaper W, Oskam J, Atasever B, Voskuil M, Ubbink D, Schirmer SH, Buschmann I, Bode C, Buschmann EE. Design of the START-trial: STimulation of ARTeriogenesis using subcutaneous application of GM-CSF as a new treatment for peripheral vascular disease. A randomized, double-blind, placebo-controlled trial. Vasc Med 2003;8:191–196.

32.Jaap AJ, Shore AC, Stockman AJ, Tooke JE. Skin capillary density in subjects with impaired glucose tolerance and patients with type 2 diabetes. Diabetes Med 1996;13:160– 164.

33.Jaap AJ, Pym CA, Seamark C, Shore AC, Tooke JE. Microvascular function in type 2 (non-insulin-dependent) diabetes: improved vasodilation after one year of good glycaemic control. Diabetes Med 1995;12:1086–1091.

34.Jaap AJ, Tooke JE. Pathophysiology of microvascular disease in non-insulin-dependent diabetes. Clin Sci (London) 1995; 89:3–12.

35.Sandeman DD, Shore AC, Tooke JE. Relation of skin capillary pressure in patients with insulin-dependent diabetes mellitus to complications and metabolic control. N Engl J Med 1992;327: 760–764.

36.Williams SB, Cusco JA, Roddy MA, Johnstone MT, Creager MA. Impaired nitric oxide-mediated vasodilation in patients with non-insulin-dependent diabetes mellitus. J Am Coll Cardiol 1996;27:567–574.

37.Stehouwer CD, Fischer HR, van Kuijk AW, Polak BC, Donker AJ. Endothelial dysfunction precedes development of microalbuminuria in IDDM. Diabetes 1995;44:561–564.

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39.Hamdy O, Ledbury S, Mullooly C, Jarema C, Porter S, Ovalle K, Moussa A, Caselli A, Caballero AE, Economides PA, Veves A, Horton ES. Lifestyle modification improves endothelial function in obese subjects with the insulin resistance syndrome. Diabetes Care 2003;26:2119–2125.

40.Economides PA, Caselli A, Zuo CS, Sparks C, Khaodhiar L, Katsilambros N, Horton ES, Veves A. Kidney oxygenation during water diuresis and endothelial function in patients with type 2 diabetes and subjects at risk to develop diabetes. Metabolism 2004;53:222–227.

41.Caballero AE, Saouaf R, Lim SC, Hamdy O, Abou-Elenin K, O’Connor C, Logerfo FW, Horton ES, Veves A. The effects of troglitazone, an insulin-sensitizing agent, on the endothelial function in early and late type 2 diabetes: a placebo-controlled randomized clinical trial. Metabolism 2003;52: 173–180.

42.Speight EL, Essex TJ, Farr PM. The study of plaques of psoriasis using a scanning laser-Doppler velocimeter. Br J Dermatol 1993;128:519–524.

43.Speight EL, Farr PM. Calcipotriol improves the response of psoriasis to PUVA. Br J Dermatol 1994;130:79–82.

44.Stucker M, Hoffmann M, Memmel U, von Bormann C, Hoffmann K, Altmeyer P. [In vivo differentiation of pigmented skin tumors with laser Doppler perfusion imaging]. Hautarzt 2002;53:244–249.

45.Stucker M, Horstmann I, Nuchel C, Rochling A, Hoffmann K, Altmeyer P. Blood flow compared in benign melanocytic naevi, malignant melanomas and basal cell carcinomas. Clin Exp Dermatol 1999;24:107–111.

46.Niazi ZB, Essex TJ, Papini R, Scott D, McLean NR, Black MJ. New laser Doppler scanner, a valuable adjunct in burn depth assessment. Burns 1993;19:485–489.

47.Park DH, Hwang JW, Jang KS, Han DG, Ahn KY, Baik BS. Use of laser Doppler flowmetry for estimation of the depth of burns. Plast Reconstr Surg 1998;101:1516–1123.

48.Pape SA, Skouras CA, Byrne PO. An audit of the use of laser Doppler imaging (LDI) in the assessment of burns of intermediate depth. Burns 2001;27:233–239.

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50.Gschwandtner ME, Koppensteiner R, Maca T, Minar E, Schneider B, Schnurer G, Ehringer H. Spontaneous laser doppler flux distribution in ischemic ulcers and the effect of prostanoids: a crossover study comparing the acute action of prostaglandin E1 and iloprost vs saline. Microvasc Res 1996;51: 29–38.

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See also BLOOD RHEOLOGY; HEMODYNAMICS.

CYSTIC FIBROSIS SWEAT TEST

WARREN J. WARWICK

University of Minnesota

Minneapolis, Minnesota

INTRODUCTION

Cystic fibrosis (CF, cystic fibrosis of the pancreas, mucovisidosis) is an autosomal recessive genetic clinical condition that occurs in 1 in 2500 Caucasian newborn infants. The frequency ranges from 1:200 in genetically isolated populations to 1:30,000 in certain ethnic and racial groups.

The CF mutations occur in a gene located in region q31.2 on the long (q) arm of human chromosome 7. The gene contains 250,000 base (amino acids) pairs in 27 exons. These base pairs code for a 1480 amino acid molecule, which serves both as a transmembrane channel for the transfer of water and salt and other substances across the cell membrane and has been given the name: cystic fibrosis transmembrane regulator (CFTR) protein.

One mutation, the DF508 mutation, accounts for twothirds of the mutations in Caucasian populations. Over 1338 mutations of the CFTR gene have been found in patients with one or more of the CF associated diseases although > 100 mutations have been found in patients with as yet none of the CF associated diseases. A few patients have been found with increased numbers of CF associated diseases, but with no mutations in the CFTR gene (1). These patients are presumed to have similar risk factors caused by other, as yet not identified, transmembrane channels. Such mutations or combinations of gene

mutations, even in a large population, occur very rarely by chance (2). The most complete source of information about the CFTR gene is in the Cystic Fibrosis Mutation Database at http://www.genet.sickkids.on.ca/cftr/.

The CFTR protein controls sweat gland chloride ion transport as well as regulating other chloride secretory channels. This abnormality of chloride transport, which has been observed with almost all mutations, is an increased excretion of salt in the sweat. This elevation of chloride in sweat is seen in > 98% of Caucasian CF patients. The clinical heterogeneity of CFTR expression is less obvious in the lung and other organs producing the diversity of clinical expression (phenotypes) of CF (3).

The reduced salt absorption in cystic fibrosis sweat glands is due primarily to poor chloride absorption with secondarily poor sodium absorption (4). A cyclic adenosine monophosphate (cAMP)-mediated sweating rate test has been developed that demonstrates a quantitative discrimination of CFTR function. This function may help distinguish between homozygous CF, CF carrier, and non-CF (5).

The measurement of the increased chloride in the sweat is the most reliable diagnostic test for the presence of two mutations of the CFTR mutation. This excessive sweat chloride is found in > 98% of patients with the genetic potential to develop the clinical diseases associated with these CFTR mutations.

HISTORY

Although the folklore of many Caucasian peoples record that an infant who tastes salty will die young, the first modern confirmation of the excessive amount of salt in the sweat of children with CF was of seven patients with cystic fibrosis admitted to a New York City hospital with heat prostration

(6). Although five other children also had heat prostration the unusual association with cystic fibrosis of the pancreas was investigated by di SantAgnese et al. (7) who found abnormal levels of sodium, chloride, and potassium in the sweat of these children. Their crude techniques for collection of sweat were inconsistently applied and produced nonstandard sweat samples for analysis, still all analyses consistently showed an increased amount of ions in the sweat.

Normal values were soon developed for children and adults without CF and parents of children with CF were shown to have slight, but significantly elevated, chloride and sodium in their sweat (8,9). The large amount of sweat required for these tests was obtained by sweating an arm or a leg of an adult or large child and required whole body sweating of small children. Such total body sweating led to some deaths due to heat exhaustion of some infants with genetic CF.

DEVELOPMENT OF A GOLD STANDARD

This uniform finding that children with CF of the pancreas have 10 times the amount of salt in their sweat created the need for a simple, cheap, rapid, and precise way to determine the salt content of sweat. Six years later L.C. Gibson, working in RE Cooke’s laboratory, developed a technology

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and technique (10) for sweat stimulation that (1) is simple and quick, (2) almost always produces a sufficient (> 70 mg) amount of sweat, (4) has virtually zero risk of injury with equipment, and (5) could be built by any electrician associated with a hospital laboratory (11). Soon, laboratories worldwide adopted this technology and showed that > 95% of normal patients had sweat chlorides < 30 mmol/L, whereas almost all patients with CF had sweat chloride values > 60 mmol/L. This method has been named the Gibson–Cooke Sweat Test (GCST) honoring the innovators and the Quantitative Pilocarpine Iontophoresis Test (QPIT) identifying and focusing on the key elements of the technology. Both names may be regarded as interchangeable and equally appropriate abbreviations of the excessively long ‘‘Quantitative Gibson–Cooke Pilocarpine Iontophoresis Sweat Test’’ (GCST/QPIT).

Over the subsequent 40 years the (GCST/QPIT) has been validated and confirmed as the only to be trusted sweat test technology for the laboratory diagnosis of CF. This GOLD Standard label has persisted despite the extreme care that must be taken to assure accurate results. The basis for that consensus is founded on three factors; (1) a known amount of sweat, (2) the chloride concentration provides the greatest discrimination, and (3) the arithmetic difference between unit measurements provides the same visual distance throughout the physiological clinical range of sweat ion concentrations.

Because of the many sources of technical error that exist with the GCST/QPIT, efforts have been made to develop alternative technology that are easier to do, have fewer risks of errors, and are simple enough to be used in general hospitals, clinics, and even physicians offices. So far, because of the potential for missed diagnoses, the best of these have only reached approval and are recommended for use only as screening tests (12).

The GCST/QPIT is the most valuable laboratory test for the diagnosis of CF. The primary indication for the GCST/ QPIT is the presence of one or more of the common CF associated diseases, which include malabsorption, failure to thrive, recurrent pulmonary infection, chronic obstructive lung disease, nasal polyps, chronic sinusitis, male infertility, gallstones, unexplained cirrhosis, arthritis, diabetes, bleeding due to vitamin K deficiency, asthma, rectal prolapse, intussusception, meconium ileus, night blindness due to vitamin A deficiency, hyponatremia, bowel obstruction, volvulus, acute pancreatitis, and the child who tastes salty. Other required reasons for performing the sweat test include immediate family history (to include first cousins) of a patient having CF, a positive or suspicious newborn screening for cystic fibrosis, and the request of the parent for a sweat test.

The purity of the GCST/QPIT technology has been and continues to be maintained (13) by many CF Center Directors, national and international organizations, including Cystic Fibrosis Foundations, Directors of Clinical Laboratories, Cystic Fibrosis Center Directors, medical specialties including Pediatricians, Pulmonologists, Gastroenterologists, Clinical Biochemists and Pathologists. Their concerns to keep the GCST/QPIT technology pure and accurate have been the object of many papers and publications with the most complete being the 97 page Guidelines

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for the Performance of the Sweat Test for the Investigation of Cystic Fibrosis in the UK, Report from the Multi-Dis- ciplinary Working Group with Representation from the Association of Clinical Biochemists, British Paediatric Respiratory Society, British Thoracic Society, Cystic Fibrosis Trust, Royal College of Paediatrics & Child Health, Royal College of Pathologists. UK National External Quality Assessment Schemes. This guideline has been formally appraised and endorsed by The Royal College of Paediatric and Child Health (http://www.acb.org.uk/Guidelines/ sweat.htm) November 2003.

THE STATE OF THE ART OF SWEAT TESTS

While CF Center Directors are focusing on the precision of this test, as it is the most constant abnormality identifying CF, there have been many attempts to help the physician in practice to screen patients with some of the classic CF symptoms, and so to avoid the need, cost, and inconvenience of referring such patients to CF Centers for the approved GCST/QPIT best test.

Harry Shwachman, doyen of CF Center Directors, made the first screening test using agar plates filled with silver chromate (14). He used these agar plates on ward rounds and in clinics by placing the child’s hand firmly on the silver chromate filled agar. Any salt on the hands would produce a strong white silver chloride image of the hand. This dramatic and immediately apparent test could give a false negative test if a CF patient had newly washed hands and could give a false positive test when a non-CF patient had been eating salty hand food such as potato chips. Because patients with positive screening tests and negative screening tests in patients who had classic CF related diseases still needed to be referred to the sweat test laboratory for the GCST/QPIT, this screening test is no longer used.

Over the 40 years the physiological basis for the GCST/ QPIT has been known, efforts to develop a screening test that might be accepted as being as accurate as the GCST/ QPIT, which might be done in a non-CF Center Laboratory, have received little support from CF physicians and Foundations.

Never-the-less three such tests have reached the attention of enough CF Center Directors to warrant a multi-CF Center study of their efficiency. These tests were (1) the CF Indicator System (15) a compact configuration of manufactured electrodes that dispense pilocarpine and a manufactured chloride sensor patch that collected a standard amount of sweat and was read as ‘‘normal’’, ‘‘CF’’ or ‘‘questionable’’ (16); (2) the chloride electrode in situ measurement of pCl after sweat stimulation by pilocarpine iontophoresis (17); and (3) the Macroduct system that used pilocarpine iontophoresis with visible collection of sweat in a plastic tube followed with conductivity or osmolarity measurements to match with a comparable concentration of sodium chloride (17).

The conclusions of the Cystic Foundation were that in the hands of community laboratories the potential for errors was so large that these tests should be considered only as screening sweat tests and that diagnostic GCST/

QPIT tests for diagnosis should be done only at CF Center sweat testing laboratories.

Never-the-less the pressure to generalize sweat testing so CF diagnosis could be done before referral to CF Centers continued to be a powerful pressure for improving these three technologies. The CF Indicator was redesigned into a new integrated system that has been built, patented, and tested in one study (18). This study showed that compared to the GCST/QPIT the Quantum Patch (19) had equal sensitivity (94%) and specificity (99%), but differed in rate of failed tests, 1% for the Quantum Patch as compared to 15% for the GCST/QPIT. The Quantum test was faster, calculated the required amounts of sweat (3–10 mg) compared to the extra step of weighing and the larger amount of sweat (70 mg) required for the GCST/QPIT, was simpler to perform, required less equipment, less expensive equipment, and was less operator dependent. As of February 2005 the Quantum Patch technology, which includes a stimulator with disposable electrode, the Quantum Patch, and a scanner that simultaneous scans the patch and calculates the weight of the sweat and the chloride concentration, has not yet received FDA approval. The manufacturing standardization of the Quantum Patch test eliminates the substantial requirements for laboratory control and supervision and shortens the time for analysis to < 5 min compared to the GCST/QPIT required time of > 1 h. The simplification and the brief time needed may make this a diagnostic sweat test that could be done in a doctor’s office or clinic. This rapid, simple, and quantitative pilocarpine iontophoresis sweat test has yet to be vetted by CF Scientists who have no financial interest in the product.

The chloride electrode (20–23) measures the pCl providing a true and immediate measurement of the chloride content of the sweat, but without a measure of the weight of the sweat. This technique might be resuscitated if an authoritative organization would mandate strict and inviolate guidelines for performance. If this could happen and if the CF Physicians and Organizations would accept such in situ measurements without knowing the weight of sweat or the rate of sweating, then diagnostic results could be known immediately. Given the strictness of the mandated strict and inviolate guidelines for performance this test might still be confined to CF Centers and other authorized Sweat Test Laboratories.

The Macroduct system is able to measure two abnormalities of the mixed-ion content of cystic fibrosis sweat. Both osmolarity and conductivity measure the ignored abnormality, the increased amount of all electrolytes in the sweat, and so have ignored what might be proven as an equal or better way to discriminate CF from non-CF subjects. Unfortunately, instead of adding new science to the study of this CF sweat abnormality, the manufacturers have reported the conductivity, or the osmolarity, of the sweat chloride concentration of a salt solution with that amount of conductivity or osmolarity. While such adjusted sweat chloride values can be used to discriminate between CF and non-CF subjects (24), the numbers are nonphysiological and so are offensive to and rejected by CF Center Directors and Sweat Test Laboratory Directors. In addition, the sweat chloride numbers confuse some practicing physicians who misdiagnosis some patients as carriers or

even patients when the MacroDuct pseudosweat chloride numbers are in the GCST/QPIT intermediate or low CF ranges. Fortunately, two groups (25,26) have demonstrated that either conductivity or osmolarity can be used as new demonstrations of diagnostic alterations of sweat gland secretion. Such efforts should be encouraged.

THE POTENTIAL FOR A GCST/QPIT SUCCESSOR

The excellent work done so far has not closed the possibility that an improved sweat test cannot be developed (27). In a multistep procedure, nonstimulated sweat was collected for 10 min from the surfaces of either thumb for 10 min, while the rate of sweating was measured from the other thumb. The thumb collections are reversed and repeated a second time. Calculations ‘‘estimate the chloride concentration by dividing the amount of chloride per unit area of one finger by the amount of sweat per unit area of the other finger’’. The sweat chloride values were similar to GCST/ QPIT sweat chloride values. Most laboratories will find this technique more tedious and filled with potential for errors than the GCST/QPIT method which, because of its reliability, accuracy, and dependability, remains the gold standard for the diagnosis of CF. The QPIT/GCST is and has been the worldwide standard because of its accuracy. However, because of the many sources of potential error and the detailed steps that are needed to ensure that accuracy the search will continue to find an equally accurate replacement.

At this time there are three candidates.

1.The Quantum Patch has been designed to have ‘‘all’’ sources of errors from the preparation of iontophoresis solutions, stimulation time, collection quantities, and computerized measurement of sweat weight and chloride concentration standardized by the manufacturer to give virtually 100% successful tests with sensitivity and specificity equal to the GCST/QPIT. Unfortunately, at this time the only published paper was published by the developers who have a financial interest in the product and the product is awaiting FDA 510K approval. If this product is well vetted by CF Centers and Sweat Test Laboratories it will be worthy of large scale tests by others. The Quantum Patch meets all the QPIT standards.

2.The chloride electrode test has had mixed reviews in the literature. It has the potential to deliver chloride concentrations within minutes after pilocarpine iontophoresis. However, because of inadequate attention to the details of performance the technology has done poorly in many hands. It has one major defect in that the weight of sweat or the rate of sweating are unknown. If standards for maintenance of equipment and of performing the test were to be simplified and standardized to CF Center and Sweat Test Laboratories requirements this technology could supersede the GCST/QPIT. Unfortunately, at this time there is little interest in such development. The chloride electrode test does not meet all of the QPIT standards.

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3.The MacroDuct system is the only system currently manufactured, supported, and maintained. It is reliable and has acquired some supporters among CF Center Directors despite that it measures only conductivity or osmolarity which, with rare exceptions, is converted to the chloride content of NaCl solutions of similar conductivity or osmolarity. The novel collection system is excellent and provides the potential for quantifying both sweat rate and weight. The MacroDuct system is the only method that takes advantage of the initial description of the sweat abnormality, the increased sweat concentrations of Na, K, and Cl, but loses that advantage by reporting the mixed ion content of sweat by specimen, conductivity, or osmolarity, results equal to a solution of NaCl. The manufacturers and the CF Centers that favor this test, with two published exceptions, have failed to develop ion content as an alternative and as an equal or more specific abnormality characterizing CF. The pseudochloride concentration carries the risk of misdiagnosis, therefore this test will probably continue to carry the label of screening test in most CF physician’s minds. New science, study of total ion content of sweat, might make this method a suitable replacement for the GCST/QPIT. There is no indication that the manufacturers are willing to make such an effort. As marketed and at this writing the MacroDuct system does not meet all of the QPIT standards.

BIBLIOGRAPHY

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2.Boyle MP. Nonclassic cystic fibrosis and CFTR related diseases. Curr Opin Pulm Med 2003;9(6):498–503.

3.Jiang Q, Engelhardt JF. Cellular heterogeneity of CFTR expression and function in the lung: implications for gene therapy of cystic fibrosis. Eur J Hum Genet 1998;6:12–31.

4.Reddy MM, Light MJ, Quinton PM. Activation of the epithelial Na(þ) channel (ENaC) requires CFTR CI( ) channel function. Nature (London) 1999;402:301–304.

5.Callen A, Diener-West M, Zeitlin PL, Rubenstein RC. A simplified cyclic adenosine monophosphate-mediated sweat rate test for quantitative measure of cystic fibrosis transmembrane regulator (CFTR) function. J Pediat 2000;137:849–855.

6.Kessler WR, Andersen DH. Heat prostration in fibrocystic disease of the pancreas and other conditions. Pediatrics 1951;8:648–56.

7.di Sant’ Agnese PA, Darling RC, Perera GA, et al. Abnormal electrolyte composition of sweat in cystic fibrosis of the pancreas: clinical implications and relationship to the disease. Pediatrics 1953;12:549–563.

8.Darling RC, diSant’Agnese PA, Perera GA, Andersen DH. Electrolyte abnormalities of sweat in fibrocystic disease of pancreas. J Med Sci 1953;225:67–70.

9.Di Sant’ Agnese PA, Darling RC, Perera GA, et al.Abnormal electrolyte composition of sweat in cystic fibrosis of the pancreas: clinical implications and relationship to the disease. Pediatrics 1953;12:549–563.

10.Gibson LE, Cooke RE. A test for concentration of electrolytes in sweat in cystic fibrosis of the pancreas utilizing pilocarpine electrophoresis. Pediatrics 1959;23:545–549.