Measuring Chloride Concentration in Sweat Collected on Gauze or Filter Paper

 

Reagents and supplies:

(1) acid diluent (glacial acetic acid plus concentrated nitric acid)

(2) gelatin reagent with pH indicator, usually with pink indicating acidity

(3) chloride calibrators, 1.0 mmol/L and 50 mmol/L

(4) low and high chloride controls

(5) preweighed gauze or filter paper

(6) paraffin wax film

(7) clean, dry vials

(8) powder free gloves

 

Instrumentation:

(1) sweat collection instrumentation for providing pilocarpine iontophoresis

(2) chloridometer (for coulometric titration)

(3) accurate analytical balance sensitive to 0.0001 g

 

Specimen collection:

(1) Using preweighed gauze or filter paper is placed over the site of the positive electrode and covered with paraffin film to prevent evaporation.

(2) For complete details on specimen collection, refer to the NCCLS document.

(3) After sweat collection, the gauze or filter paper is removed and reweighed.

(4) The increase in weight corresponds to the volume of the sweat collected. The relative density of sweat ranges from 1.001 to 1.008.

(5) The sweat is then eluted from the gauze or filter paper using a volume of Type I water.

 

Test vial types, prepared in duplicate:

(1) blank vial

(2) gauze or filter paper blank vial

(3) calibrator vial

(4) test vial

(5) low control vial

(6) high control vial

 

Each vial contains:

(1) 3 mL of acid diluent

(2) 1 mL of fluid based on vial type:

(2a) Type I water for blank vial

(2b) test eluate for patient vial

(2c) control eluate for control vials

(2d) gauze or filter paper eluate in gauze or paper blank vial

(2e) calibrator solution for calibrator vial

(3) 4 drops of gelatin reagent

 

Chloride measurement:

(1) Chloride is measured on each test vial type prepared in duplicate.

(2) The result for each vial type is the average of the two readings.

 

Quality Control:

(1) amount of sweat collected: At least 0.075 grams of sweat should be collected when 2x2 inch gauze is used.

(2) weight of control vial: Usually 100 µL of control is added to the gauze in the control vial. The difference in weight of the vial after addition of the control solution from that before should be 0.1 gram +/- 2%. If the weight differs significantly from this, then the pipet and scale should be checked.

(3) color after addition of gelatin reagent: The solution's acidity is indicated by a pink color after 4 drops of gelatin reagent are added to the diluted sample. Do not titrate if the pink color is not present.

(4) controls: The low and high chloride controls should give results comparable to formulated concentrations.

(5) technique: The operator must ensure that no artifact is introduced during the procedure. The gauze or filter paper must not be handled without gloves and the gloves should be powder free. Paraffin wax film is used to minimize evaporation during the specimen collection period.

 

sweat chloride for patient in mmol/L =

= A * B * (chloride concentration of calibrator used)

 

where

• A = ((chloride in test vial in mmol/L) - (chloride in gauze or filter paper blank vial)) / ((chloride in calibrator vial) - (chloride in blank vial))

• B = ((mL of dilutent used to elute gauze or filter paper) + (grams of sweat collected or control added)) / (grams of sweat collected or control)

 

chloride concentration for controls in mmol/L

• unclear from NCCLS documentation how this is calculated

 

Interpretation:

• A chloride concentration > 60 mmol/L is consistent with the diagnosis of cystic fibrosis.

• A chloride concentrations from 40-60 mmol/L is considered borderline.

• A chloride concentrations < 40 mmol/L is negative.

• Some unaffected adults may have chloride concentrations > 60 mmol/L, and several disorders other than cystic fibrosis are associated with an increased sweat electrolyte concentration.

• The diagnosis of cystic fibrosis requires more than one chloride determination and should be based on the patient's clinical picture by a physician knowledgeable about cystic fibrosis.