August in Texas is a good time to write a blog post about sweat. In this case though, I’m going to specifically talk about testing collected sweat samples for chloride concentration. Sweat chloride concentrations are measured in people who are suspected of having Cystic Fibrosis (CF). Because CF has classically been considered a disease of childhood, sweat chloride testing is performed almost exclusively in pediatric institutions.
CF is a disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. This is a large gene which codes for a large, transmembrane protein that acts as a chloride channel. More than 1500 mutations have been detected in the CFTR gene, not all of which are known to cause disease. Thus, even though the full gene has been sequenced, CF remains a diagnosis which is made by a combination of the presence of characteristic clinical features, or history of CF in sibling, or a positive newborn screen, PLUS identification of a disease-causing mutation in the gene or protein or laboratory evidence of chloride channel malfunction such as an elevated sweat chloride level.
Collecting a sweat sample for testing is an interesting manual process. The first step involves stimulating the sweat glands to produce sweat. This is accomplished by a process called iontophoresis, in which a sweat-gland-stimulating compound called pilocarpine is driven into the skin using a small electrical current between a set of electrodes applied to the skin. After a 5 minute stimulation, the electrodes are removed, the skin is cleaned, and the sweat that is subsequently produced in that stimulated area is collected for the next 30 minutes. The collection is either via absorption of the sweat into a piece of gauze or filter paper, or by a collection device which funnels the sweat into a small plastic tube as it’s produced. The amount of sweat collected after 30 minutes is determined by weight if gauze or filter paper is used, and by volume if the tubing is used. There is a lower acceptable limit for both cases, below which the sweat collection is insufficient (QNS) and must be repeated. The process sounds simple, however collecting a sufficient quantity of sweat can be problematic, and collecting too little may cause falsely elevated results.
After collection, the amount of chloride present in the collected sample is measured. In a normal sample, the amount of chloride present is well below the measurement range of the usual chloride ion-selective electrodes found in chemistry or blood gas instruments. For this reason, the chloride concentration in a sweat sample is most commonly measured using a method called coulometric titration in which a silver electrode placed in the sample gives off silver ions during a current flow. The silver ions complex with the chloride and precipitate as silver-chloride. This reaction continues until all the free chloride is gone, at which point a timer stops. Quantification is accomplished essentially by comparing the time necessary to complex all the patient’s chloride versus the time necessary to complex a known concentration of chloride in a standard. Calculations are performed using the time and the weight or volume of the sweat collected, among other parameters.
The entire test is very manual. Collection of appropriate sweat samples requires training and practice. In general the QNS rate – how often an adequate collection is not achieved – is carefully monitored by the lab, the CF clinic and the CF Foundation which accredits the clinic. In addition, measuring the chloride in the sweat by chloridometer is not an automated process of placing the sample on an instrument and pushing a button to go. For these reasons, the CFF recommends not performing sweat testing unless you perform a minimum number per year in order to stay proficient. In this day and age of increasing automation, sweat chloride testing remains the anomalous, old fashioned test requiring significant technologist time and expertise.
-Patti Jones PhD, DABCC, FACB, is the Clinical Director of the Chemistry and Metabolic Disease Laboratories at Children’s Medical Center in Dallas, TX and a Professor of Pathology at University of Texas Southwestern Medical Center in Dallas.