This study employed the use of fifteen individuals (9 males, 6 females, ages 21-61 years, Fitzpatrick Skin Types II-III years) who all signed informed consent to participate in the study. All the participants had healthy, undisrupted inner volar forearm skin and had not taken antibiotics or used any type of potential sanitizing or exfoliating products two weeks prior to participation in the study. Both arms of each participant were used that allowed inclusion of both a test site and a control, untreated, site that allowed for a total of n=30 inner volar forearms to be examined, as shown in Figure 4. Prior to participating in the study, everyone was instructed to do their routine morning washing routine but to not use soap or scrub on their inner volar forearms to rinse the arms with warm water and to not apply any types of products to their arms for the duration of the study.
Charm Science PocketSwabs and NovaLum ATP Analyzer
For these studies, a NovaLum VX-II ATP Analyzer from Charm Sciences, Inc. [Andover, MA] was employed to measure ATP biofluorescence. The company also provides PocketSwabÒ Plus swabs. A picture of a PocketSwab is shown below in Figure 5.
PocketSwab Plus is a self-contained unit that has a sterile swab stored until use in a sterile chamber. When ready for use, the swab is withdrawn from the unit and is applied to the surface being tested using specific directions offered by the company, which includes a swabbing pattern that requires approximately sixteen square centimeters of surface while rotating the swab and applying a specific amount of pressure to the surface. The resulting contaminated swab is then inserted back into the unit, and with a twisting screw, motion is thrust through a thin foil membrane that houses an oxidizing fluid. The tip of the swab and the oxidizing fluid drop into the clear chamber at the bottom of the device. The device houses a small tablet that contains a luciferin dye that is activated by the oxidizing fluid and a luciferase enzyme pellet that has been designed to detect adenosine triphosphate (ATP). The device then fluoresces in accordance with the amount of ATP present on the swab. The resulting activated unit is placed into the NovaLum ATP analyzer shown in Figure 6. The device then measures the amount of luciferin biofluorescence and reports the values in relative fluorescent units (RFUs), which can be recorded for testing purposes.
All measurements were made at a room temperature of 22-24 °C and an RH of approximately 45-50%, and each participant equilibrated to the room for 15 minutes prior to measurements being taken. The participants were tested initially at all four inner volar forearm locations before any treatments to obtain a baseline level of ATP based on the RFUs noted from the unit for each site. Each participant was then swabbed with a cotton ball saturated with 3% hydrogen peroxide at the two test sites noted in Figure 1. The adjacent site was not swabbed and remained as an untreated control site on each arm. The participants waited five minutes, and the two sites were swabbed with hydrogen peroxide again. This sanitizing treatment was repeated a third time, and then the participants could continue their normal activities for one hour with the stipulation that they take care not to rub their arms against any solid surfaces or any other skin surfaces for the duration of the first day of the study. The participants offered both arms, which doubled the number of treatment sites, so a total of 30 arms were tested. The two arm sites were also alternated, as shown in Figure 1. Each participant was then measured using the Novalum ATP Analyzer with a single swab at each site at the following timepoints: T(1): 1 hr, T(2): 3 hrs, T(3): 6 hrs, T(4): 8 hrs and T(5): 24 hrs.
The RFUs for each timepoint were collected and averaged. A total of twelve total measurements (T(0)-T(5)) were made through the course of each individual study using both arms, and the results for each time point were averaged. The results were then compared between the test sites (H2O2-treated) and the control sites. In addition, the results at Timepoints T(1), T(2), T(3), T(4) and T(5) were normalized by dividing the average RFUs for each timepoint measurement by the averaged baseline measurement to provide normalized hourly comparisons accounting for initial variations in site ATP levels. This provided a unitless measure of normalized data for each timepoint. Using the statistical function in Microsoft Excel, the Student Paired, two-tailed T-Test was employed to determine 95% confidence (p≤0.05) for each data point.