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The diagnosis of Cystic Fibrosis: (Taken from a recent "Consensus statement" in the Journal of Pediatrics)

Cystic Fibrosis is the most common life-limiting recessive genetic disorder in Caucasians, with an incidence of 1 in 3200 newborns in the United States.' It is less common in African Americans (1 in 15,000) and in Asian Americans (1 in 31,000). The CF gene product, CF Transmembrane Conductance Regulator, functions as a cyclic adenosine monophosphate-regulated chloride channel at the apical surface of epithelial cells. Thus far, more than 500 mutations of the CFTR gene have been identified In the presence of CFTR mutations, dysfunctional epithelial transport leads to the clinical manifestations seen in patients with CF. In the United States, the diagnosis of CF is established by 1 year of age in the majority (71%)of patients. However, in 8% of patients the diagnosis is not established until after age 10 years, and the diagnosis is now being made in an increasing number of adults.

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  • It is essential to confirm or exclude the diagnosis of CF in a timely fashion and with a high degree of accuracy to avoid unnecessary testing, to provide appropriate therapeutic interventions and prognostic and genetic counseling, and to ensure access to specialized medical services. In the majority of cases, the diagnosis of CF is entertained because of the presence of one or more typical clinical features and then confirmed by demonstrating an elevated (>60 mmol/L) sweat chloride concentration Almost all patients have chronic sino-pulmonary disease and, in post-pubertal men, obstructive azoospermia. Approximately 85% to 90% of all patients have ex pancreatic insufficiency.
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    In recent years the ability to detect mutations and to measure transpeptide bioelectric properties has greatly extended the CF clinical spectrum. In approximately 2% of patients, there is an equivocal phenotype, which consists of of sinopulmonary disease, pancreatic insufficiency, and either borderline (40 mmol/L) or normal (<40 mmol/L) chloride concentrations. In addition there are patients in whom a single dagnostical feature (e.g., electrolyte abnormalities, pancreatitis, liver disease nusitis, or obstructive azoospermia predominates. In such cases, demonstration of CF mutations in each CFTR or the in vivo demonstration of abnormal transport across nasal epitheliui be used as diagnostic aids.

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    A CF diagnosis can also be considered in the absence of clinical features. An individual with an infected sibling has a 1 in 4 (25%) chance of having the disease. Half siblings are also at increased risk compared with the general population. ( Caucasians 1 in 112; African Americans 1 in 244 and Asian Americans 1 in 352 ) These high risks justify careful clinical monitoring and in appropriate situations, sweat testing of full and half siblings.

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    In many parts of the world, a diagnosis of CF is being established in the neonatal period with increased frequency because of the inclusions of tests for CF in newborn screening programmes. In these programmes the diagnosis is suggested by an elevated level of immunoreactive trypsinogen in the blood and then confirmed by mutational analysis or sweat testing. Although some of these infants may be free of the symptoms at the initial time of testing, it can be predicted that virtually all such patients will experience clinical manifestations of CF. Finally, because of the availability of accurate prenatal testing, an in-utero diagnosis of CF, based on the detection of two CF mutations in the fetus, is being made with appreciable frequency. Such testing is usually carried out in a family that has a previously affected child or because of the detectionof fetal echogenic bowel on routine ultrasonography. Additional cases may be diagnosed as a result of the expansion of CF carrier screening in the general population.

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    The goal of the Consensus panel was to more precisely define the criteria for a diagnosis of CF in the context of our evolving understanding of the diverse clinical features seen in patients with CF and the availability of new diagnostic procedures.

    It is the consensus of the panel that the diagnosis of CF should be based on the presence of one or more clinical features, a history of CF in a sibling, or a positive newborn evidence of a CFTR abnormality as documented by an elevated sweat chloride concentration, or identification of mutations in each CFTR gene known to cause CF or in vivo demonstration of characteristic abnormalities in ion transport across the nasal epithelium. In most cases the diagnosis of CF will be confirmed by measurement of chloride concentration in sweat after iontophoresis of pilocarpine. Sweat testing should be carried out in accordance with the guidelines of the National Committee for Clinical Laboratory Standards. It is crucial that testing be carried out by experienced personnel, using standardized methods in facilities that perform adequate numbers of tests to maintain laboratory efficiency and quality control.

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    The only acceptable procedure is the quantitative pilocarpine iontophoresis sweat test. A minimum acceptable volume is 15 µl for the Wescor Macroduct coil system, or weight 75 mg for the Gibson-Cooke procedure. Sweat must be collected during a 30-minute period to ensure an average sweat weight of more than 1gm/m2/min.

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    Alternative sweat test procedures such as direct-reading conductivity measurements or a paper-patch indicator system are associated with an increased incidence of false-positive and false-negative results and should never be used as the basis of a definitive CF diagnosis When a physician orders a sweat test, it is mandatory that he or she know the method being used, as well as reference laboratory values.

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    Analysis of Sweat Chloride by Coulometric Method

    Using the Sherwood Scientific Model 926S CHLORIDE ANALYSER

    Clinical Indication

    The sweat test is the gold standard for the diagnosis of cystic fibrosis, which is confirmed by elevations in the sweat electrolytes, principally chloride ions.

    Patient Sample

    The known weight of sweat from patients having undergone iontophoretic stimulation with pilocarpine will require elution in 10mls of distilled water before analysis.

    NB If sodium is analysed on the same sample then the diluent will be 100 ppm Lithium carbonate in distilled Water

    Calibrator

    Sherwood Multical containing 140mmol/l sodium, 100mmol/l chloride.
    Catalogue Number:47852000

    Equipment

    Sherwood Scientific Model 926S Analyser ( Part 92611005) Positive displacement pipette. Adjustable 1 -5 ml pipette.

    Reagents

    Combined acid buffer. Catalogue Number 00156608
  • Polish for silver electrodes. (Catalogue Number 00156205) Sherwood Scientific
  • Diluent prepared from (Cat 00156222) Sherwood Scientific (01223 243444)

    Procedure

    1. Switch on the instrument on and allow 15 minutes warm up to meet the stated specification.
    2. Select the '20µ1' sample volume on the instrument.
    3. Pour the Combined Acid buffer into the measuring beaker up to the etched mark and press Condition
    4. Pipette accurately 20 ul of Calibrator solution and then press Titrate. Repeat to get 2 readings within range 97-103
    5. Keeping the electrodes immersed, pipette 1 ml of sweat eluate into the beaker and depress the Titrate button. When the stirrer stops, note the reading on the display. Repeat this cycle for each test
    6. When the 'change reagents' indicator comes on, lower the beaker platform and empty the beaker contents. Rinse with distilled water and dry with tissues.
    7. Continue with fresh combined acid buffer. Recondition the electrodes and proceed with checking the calibration.
    8. When determinations are complete, remove the beaker and allow the electrodes to dry naturally.

    Results and Calculation

    Sweat chloride in mmol/l = Reading of Analyser x0.200 Divided by Weight of sweat in grammes

    Round results off to the nearest whole number and when the controls have been verified as within the acceptable range (+1- 2SD) report results to the requesting clinicians and in the laboratory computer. Abnormal results, equivocal results and patients that yield small volumes of sweat are always repeated.

    Reference Range

    Sweat chloride:
    Less than 40 mmol/l40-60mmol/l greater than 60 mmol/l
    NEGATIVEEQUIVOCALConsistent with CF. POSITIVE

    Sweat chloride concentrations should be interpreted by a physician knowledgeable about CF with regard to the clinical presentation, patient's age and the knowledge that rare CFTR mutations are associated with borderline or negative sweat chloride results.

    A sweat chloride concentration of more than 60 mmol/L is consistent with the diagnosis of CF .Some data suggests that in infants younger than 3 months of age, a sweat chloride concentration of more than 40 mmol/L is highly suggestive of a diagnosis of CF. The diagnosis of CF should be made only if there is an elevated sweat chloride concentration (>60 mmol/L) on two separate occasions in a patient with one or more clinical features consistent with the CF phenotype or a history of CF in a sibling.

    Because sweat sodium concentrations of 60 to 80 mmol/L can be seen in individuals with diseases other than CF measurement of sodium alone is not recommended.

    However, in some cases, especially those with borderline sweat results, measurement of both sodium chloride concentrations can be helpful In patients with CF, both analytes should be proportionately elevated (within 15 mmol/L). and the chloride/sodium ratio is almost always greater than 1.0 A sweat chloride concentration of me than 160 mmol/L is physiologically impossible and suggests an error in collection or analysis. Tests with such results should be repeated.

    Sherwood Scientific manufactures the Model 420 Flame Photometer and has a protocol which allows for the measurement of chloride and sodium from a single Sweat Sample